*4.7. Data Analysis of Small RNA Sequencing*

The expression level of miRNAs was compared between spot and non-spot tissues to identify differentially expressed miRNAs (DEMs). The differentially expressed miRNA between spot and non-spot tissues were identified and filtered with the R package DESeq2 and Poisson Distribution [38,44]. The DEMs were determined based on FDR ≤ 0.001 and the absolute value of |Log2FC| ≥ 1. The heatmap displays of the Trimmed Mean of M-values (TMM) normalized against the Fragment Per Kilobase of transcripts per Million mapped reads (FPKM) were performed created using the R package pheatmap (https://cran.r-project.org/src/contrib/Archive/pheatmap/).

The potential target genes by differentially expressed miRNAs were predicted and analyzed with 2 different software, including psRobot [45] and Targetfinder [46]. In order to increase the level of confidence and get more reliable results, we selected only those binding sites that were predicted by both of two softwares.

All protein-coding target genes regulated by DEMs were annotated against the KEGG databases. The KEGG enrichment analysis for the target genes of the DEMs was performed by Fisher's exact test (P-values) in Blast2GO pipeline, and P-values were used to conduct multiple test correction by FDR. KEGG terms with FDR< 0.05 were considered to be significantly enriched.

### *4.8. Verification of miRNA Expression Levels by qPCR*

Two miRNA targets on key regulate gene transcription factor *PeMYB11* and *PeMYB7* were chosen for qRT-PCR analyses. The qRT-PCR analyses were performed using SYBR Premix Ex TaqTM II (Tli RNaseH Plus) (Takara, Dalian, China) according to the manufacturer's instructions with the following reaction conditions: Denaturation at 95 ◦C for 30 s and 40 cycles of amplification (95 ◦C for 5 s, 60 ◦C for 30 s). The U6 gene was used as an internal control for normalization. Relative expression levels of target genes were calculated using the 2−ΔΔ*C*<sup>t</sup> [40] against the internal control. The specific stem-ring primers were designed according to the miRNA qPCR primer design method [47], showed in Table S8. Experiments were performed with three independent biological replicates and three technical replicates.

**Supplementary Materials:** Supplementary Materials can be found at http://www.mdpi.com/1422-0067/20/17/ 4250/s1.

**Author Contributions:** Conceptualization, J.W. and X.S.; methodology, A.Z., Z.C. and T.L.; software, H.P.; materies culture, Y.S. and X.L.; validation, A.Z., Y.Z. (Ying Zhao) and Y.Z. (Yang Zhou); writing—original draft preparation, A.Z.; writing—review and editing, J.W., W.H. and T.P.

**Funding:** This study was supported by the Hainan major science program (zdkj201815) and the National Natural Science Foundation of China (31760590; 31260488). This research was conducted at Research Center for Terrestrial Biodiversity of the South China Sea, Hainan University, Hainan, China.

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