To facilitate the functional annotation of the pepper genome, analysis of miRNAs was performed for the sequenced data from five small RNA libraries described above, representing five different tissues. Starting with a set of 5,436 plant mature miRNA sequences available in miRBase, we annotated with high confidence 176 pepper miRNAs from 64 families, of which 30 families are computationally predicted to target TFs, suggesting important roles of these miRNA families in post-transcriptional gene regulation and transcription networks consistent with previous findings.Overall design: To identify genomic regions generating small RNAs, we applied previously described analytical strategies to five small RNA libraries from different tissues. Developing roots, stems and mature leaves were collected from plants grown in soil in a green house at 22 °C with a 16 hr light cycle and harvested from plants at full-bloom stage. Mature plants were harvested for fully open flowers. Additional flowers were allowed to self-pollinate and fruit was harvested in the breaker stage (thirty days after pollination when the fruit was turning red). Total RNA for different tissues was isolated from the frozen root samples by using the Trizol Reagent (Invitrogen) according to manufacturer’s instructions, and libraries were constructed using the Small RNA Sample Prep Kit (Illumina, San Diego, CA) as previously described, then sequenced on an Illumina HiSeq2000 system.