2.5. Prediction and Identification of miRNA–mRNA Regulatory Modules

The target genes of differentially expressed miRNAs were identified by aligning mature miRNA sequences with published mRNA-seq sequences [41] using TargetFinder (https://github.com/carringtonlab/TargetFinder, accessed on 8 April 2018) [47], following the procedures described in previous studies [48]. The TargetFinder algorithm is based on the miRNA and mRNA complementary-pairing principle. The alignment score value in the prediction result was used as the screening standard, which was the score of the prediction result. The comparison between the miRNA and mRNA was performed, and then the complementary pairing score was given for the comparison position: a total of 1 point was given for one mismatch or deletion, and 0.5 points were given for one G:U pairing; the penalty for a mismatched G:U pairing in the core region was doubled, and the core region began at the 2nd nt and continued to the 13th nt of the miRNA (5′–3′). Finally, the results were obtained for scores less than or equal to 4. The significance of the alignment score was the degree of match between the target gene and the miRNA. The lower the score, the more complete the matching, and the more credible it was.

The GO terms and KEGG pathways of genes targeted by these differentially expressed miRNAs were annotated. The GO function and KEGG information of this species were used to perform GO function annotation (http://bioinfo.cau.edu.cn/agriGO/, accessed on 1 September 2018) and KEGG signaling pathway annotation (https://www.kegg.jp/kegg/pathway.html, accessed on 1 September 2018) for the target genes of differentially expressed miRNAs. Fisher’s exact hypothesis test was performed on the GO and KEGG pathways of the targeted genes, and the enrichment analysis of each GO term and KEGG pathway was separately performed. According to the prediction results for the targeted genes of the differentially expressed miRNAs, significance differences in the GO and KEGG information were defined with a default threshold, p < 0.05.