Chi, M., Liu, C., Su, Y., Tong, Y. and Liu, H. 2015. Bioinformatic prediction of upstream microRNAs of PPO and novel microRNAs in potato. Can. J. Plant Sci. 95: 871-877. MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that play roles in many biological processes of plants. This study aimed to identify novel miRNAs and miRNAs targeting polyphenol oxidase (PPO) in potato. Small RNA-seq data (GSE32471 and GSE52599) including sequencing data of flower, leaf, stem, root, stolon and tuber tissue of potato were downloaded from Gene Expression Omnibus. After quality control and data cleaning of the raw data, the clean data were then mapped to Rfam to screen the reads corresponding to miRNA rather than other types of small RNA by Bowtie. Furthermore, the screened high-quality reads were mapped to known miRNAs in miRBase to identify and predict the novel miRNAs by miRDeep2. Finally, target gene prediction was performed for all identified miRNAs using psRNATarget and their roles in stress responses and brown spot of potato tubers through PPO genes were analyzed. A total of 18 novel potato miRNAs were identified and all of them had their specific expression patterns in different tissues. Targets prediction showed that some novel miRNAs (e.g., ST4.03ch03_9018, ST4.03ch05_15199 and ST4.03ch11_31208) could regulate the expression of potato resistance genes. Moreover, eight known miRNAs were found to target 3 PPO encoding genes, while they expressed at a low level in tuber tissue. Novel miRNAs might be associated with stress resistance of potato, and upstream miRNAs of PPO encoding genes might be important in suppression of potato brown spot.