*2.6. Data Analysis of Small RNA Sequencing*

Four sRNA libraries were constructed and 23,352,936, 24,896,769, 26,605,438, 25,926,370 high-quality tag were obtained, respectively (Table S5). The sequencing raw data has been deposited into the Short Reads Archive (SRA) database under the accession number SRP161646. These clean tags were mapped to reference genome of *Phalaenopsis equestris* and other sRNA databases. The average gene mapping ratio of each sample was 77.38% (Table S6).

A total of 1552 DEMs were detected in pairwise comparison with 676 downregulated and 876 upregulated (Non-spot-VS-Spot) (Figure 5). miRNA target gene prediction resulted in 1396 common unigenes between TargetFinder (http://http://targetfinder.org/) and psRobot (http://http://omicslab.genetics.ac.cn/psRobot/), 1648 by TargetFinder and 7652 by psRobot, and totally 1307 target unigenes were aligned to KEGG database. However, there were no DEMs were found targeting on anthocyanin biosynthesis structure gene. As for the regulator genes, we found totally 12 microRNAs targeting on 10 significant different expressed MYB, *bHLH* or *WRKY* unigenes (Table 3). However, only *PeMYB7* (PEQU\_03393), *PeMYB11* (PEQU\_10361, PEQU\_10362) were significantly upregulated in these TF families according to the transcriptomes data, and these microRNAs belonged to miR156 or miR858 families.

**Figure 5.** Volcano plot of DEMs. X axis represents log2 transformed fold change. Y axis represents −log10 transformed significance. Red points represent upregulated DEMs. Blue points represent downregulated DEMs. Gray points represent non-DEMs.


**Table 3.** Putative miRNAs targeting on *MYB*, *bHLH* or *WRKY* unigenes.

#### *2.7. qPCR of Key Structural Genes, Regulate Genes and miRNA*

The expression levels of 10 anthocyanin genes and 4 transcriptor genes between the non-spot and spot areas were verified by qPCR. The results showed the genes *Pe4CL2*, *PeANS*, *PeF3H*, *PeF3H1*, *PeF3 H* and *PeMYB7*, *PeMYB11* presented significantly higher expression levels in spot areas than the non-spot areas (Figure 6), which was generally consistent with the results of the transcriptome data (Table 1).

**Figure 6.** The expression level of structure gene and regulate gene Associated with Anthocyanin Biosynthesis, \* represent there are significant differences between spot and non-spot area; \*\* represent there are extremely significant differences; red: spot area, blue: non-spot area.

The expression of microRNA of RNA miR858, miR156g were also analyzed by qPCR between the non-spot and spot areas (Figure 7). The results showed that they presented higher expression levels in non-spot areas, which means their targeting genes, *PeMYB7* and *PeMYB11*, having less expression levels in the non-spot areas.

**Figure 7.** The expression level of mtr-miR156g-3p and miR858. \*\* represent an extremly significant difference (*p* < 0.01); \* represent an significant difference (*p* < 0.05).

#### **3. Discussion**
