## 5 Conclusions  
A total of 351 conserved miRNAs belonging to 216 families were identified in _Gleditsia sinensis_ pods at different developmental stages, which showed that the saponins increased gradually from May to September. A total of 36 conserved miRNAs existed specifically in legumes. A total of 708 unigenes were predicted to be the targets of 37 differentially expressed miRNAs. The targets of these differentially expressed miRNAs were involved in the regulation of terpenoid biosynthesis. To date, this is the first report of miRNA identification in _G. sinensis_ and miRNA expression profiles at different developmental stages of the pod, which provides a basis for uncovering the molecular regulation of saponin synthesis in _G. sinensis_ and new insights into the role of miRNAs in legumes.  
**Supplementary Materials:** The following are available online at: [[https://www.mdpi.com/article/10.3390/f13010108/s1](https://www.mdpi.com/article/10.3390/f13010108/s1)]([https://www.mdpi.com/article/10.3390/f13010108/s1](https://www.mdpi.com/article/10.3390/f13010108/s1)), Figure S1: The concentrations of saponins in three stages (May, July, and September) were quantified by spectrophotometry colorimetry with echinocyst acid (EA). (a) Full wavelength scanning of EA and methanol extract of pods from June to November. (b) Standard curve of echinocytic acid (EA) from 20 ug to 100 ug. Figure S2: Secondary structure of novel miRNAs (green represents the mature sequence of novel miRNA; yellow represents the corresponding star sequence of novel miRNA). Figure S3: GO enrichment of target genes in May vs. July and July vs. September. Figure S4: Top 20 KEGG enrichment pathways of target genes in July/May (a) and September/July (b) from _Gleditsia sinensis_ pods. Table S1: Saponin content and absorbance of _Gleditsia sinensis_ pods and leaves at different stages. Table S2: Known miRNAs from three developmental periods in _Gleditsia sinensis_ pods. Table S3: Known miRNA families from three periods of _Gleditsia sinensis_ pods. Table S5: Differentially expressed miRNAs from three development periods of _Gleditsia sinensis_ pods. Table S6: Predicted target number of differentially expressed miRNAs in _Gleditsia sinensis_ pods. Table S7: Predicted target number of differentially expressed miRNAs in _Gleditsia sinensis_ pods. Table S8: miRNAs involved in development and the annotation of their targets.  
[PERSON] and [PERSON] were responsible for conceptualization, bioinformatic analysis, data interpretation and drafting the manuscript. [PERSON] and [PERSON] assisted in the statistical analysis and critical evaluation of manuscripts. [PERSON] was responsible for sorting out the forms. [PERSON] and [PERSON] was responsible for supervision, project administration and funding acquisition to support this research. All authors have read and agreed to the published version of the manuscript.  
This work was supported by the National Natural Science Foundation of China (32071504 and 31670671), the Fundamental Research Funds for the Central Universities (2016 ZCQ05) and the Special Support Project of Development and Planning Division of Beijing Forestry University (2018 FGC08).  
* **Data Availability Statement:** sRNA sequencing data could be available at the NCBI SRA database ([[http://www.ncbi.nlm.nih.gov/sra](http://www.ncbi.nlm.nih.gov/sra)]([http://www.ncbi.nlm.nih.gov/sra](http://www.ncbi.nlm.nih.gov/sra)), accessed on 26 November 2021) with the accession numbers PRJNA783670, PRJNA783672, and PRJNA783674.  
**Acknowledgments:** We thank the reviewers and editors for the thoughtful comments and suggestions on the manuscript. We thank the 1 GENE company for helping with sRNA-sequencing and technical assistance.  
**Conflicts of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.