*4.6. Expression of the* D. officinale PLP\_deC *Genes in Di*ff*erent Tissues*

To obtain expression data for *D. o*ffi*cinale*, we searched the NCBI SRA database (PRJNA348403) for RNA-sequence data from different tissues [27] (Additional file: Table S2). The raw data were stripped of adapters and low-quality reads (and bases) and then rRNA and virus reads were filtered out by using Trimmomatic software with the default parameters. The clean reads were aligned to the *D. o*ffi*cinale* genome using Hisat2 [43] with the options -dta and -no-unal. The aligned outputs were converted from SAM to BAM format by using SAMtools [44]. Then, the Stringtie software was used to estimate the transcript abundances with FPKM method. The heat map of the *PLP\_deC* genes expression profiles was obtained by TBtools software (https://github.com/CJ-Chen/TBtools/releases).

#### *4.7. Quantitative Fluorescence Analysis of the* PLP\_deC *Genes*

We used the CFX96 TouchTM Real-Time PCR Detection System (Singapore) to perform a quantitative fluorescence analysis of the *PLP\_deC* genes in the cDNA samples from *D. o*ffi*cinale* protocorms with three replicates. β-actin [45–47] was used as an internal reference, the relative expression levels of genes were calculated using the 2−ΔΔCT method [48], and the primers were designed using Primer Premier 5.0 software (Table S3). Each reaction contained the following: 8 μL of SYBR Premix Ex Taq II (2x), 2 μL of template cDNA, 1 μL of forward and reverse primers, and addition of ddH2O to a final volume of 20 μL. The reaction conditions were as follows: 95 ◦C for 3 min followed by 40 cycles of 95 ◦C for 10 s, 52 ◦C for 15 s, and 72 ◦C for 30 s.

To determine whether the differential expression was significant, the difference in the relative expression of each target gene in the different treatment groups was analyzed by Student's *t*-test in SPSS 25.0 software [49].

#### **5. Conclusions**

Overall, we conducted a comprehensive analysis of *PLP\_deC* genes in both *D. o*ffi*cinale* and *P. equestris*. Comparative analysis has shown that eight *PLP\_deC* genes from *D. o*ffi*cinale* could be divided into three subfamilies: GAD, HDC, and AAD. Most genes have an acidic pI. Purifying selection may have played a key role in the evolution of this *PLP\_deC* genes in *D. o*ffi*cinale* based on the Ka/Ks value. Among them, both *DoAAD1* and *DoAAD2* were highly expressed in the column, flower bud, and lip. Under three hormone treatments, MeJA, ABA, and SA, the *PLP\_deC* genes responded to abiotic stresses. These results provide preliminary biological information for further studies of the evolution of *PLP\_deC* genes in Orchidaceae.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/1/54/s1.

**Author Contributions:** Y.C. (Yongping Cai) and Q.J. conceived and designed the experiments; L.Z., C.J., Y.C. (Yunpeng Cao), X.C., and J.W. performed the experiments; L.Z. and C.J. analyzed the data; L.Z. wrote the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** This work was supported by the Universities Natural Science Research Project of Anhui, China (Project number KJ2016A224), the University Synergy Innovation Program of Anhui Province (Project number GXXT-2019-043), and the Major Science and Technology Project in Anhui Province (Project number 17030701031).

**Conflicts of Interest:** The authors declare no conflict of interest.
