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    Evaluation of key miRNAs during early pregnancy in Kazakh horse using RNA sequencing. Liu LingLing,Fang Chao,Sun YinZe,Liu WuJun PeerJ Background:miRNA has an important role in cell differentiation, biological development, and physiology. Milk production is an important quantitative trait in livestock and miRNA plays a role in the amount of milk produced. Methods:The role of regulatory miRNAs involved in equine milk production is not fully understood. We constructed two miRNA libraries for Kazakh horse milk production from higher-producing (H group) and lower-producing (L group) individuals, and used RNA-Seq technology to identify the differentially expressed miRNAs between the two milk phenotypes of Kazakh horses. Results:A total of 341 known and 333 novel miRNAs were detected from the H and L groups, respectively. Eighty-three differentially expressed miRNAs were identified between the H and L group s, of which 32 were known miRNAs (27 were up-regulated, five were down-regulated) and 51 were novel miRNAs (nine were up-regulated, 42 were down-regulated). A total of 2,415 genes were identified. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that these genes were annotated to mammary gland development, mammary gland morphogenesis, tissue development and PI3K-Akt signaling pathways, insulin signaling pathway and TGF-beta signaling pathway, among others. Five miRNAs (miR-199a-3p, miR143, miR145, miR221, miR486-5p) were identified as affecting horse milk production and these five miRNAs were validated using qRT-PCR. Conclusions:We described a methodology for the transcriptome-wide profiling of miRNAs in milk, which may help the design of new intervention strategies to improve the milk yield of Kazakh horses. 10.7717/peerj.10796