Identification and Functional Analysis of MicroRNAs and Their Target Genes in Reverse Thermosensitive Genic Male Sterility of Eggplant

Abstract

Thermosensitive genic male sterile (TGMS) lines are the core of two-line hybrid systems. MicroRNAs (miRNAs) play critical roles in plant growth and development. However, knowledge of regulation of anther development by miRNAs in TGMS eggplant (Solanum melongena) is largely unexplored. To investigate the mechanism underlying miRNA regulation of male sterility, we employed high-throughput small RNA sequencing in anther samples from the reverse TGMS line 05ms and the temperature-insensitive line S63 in eggplant, under high temperature and low temperature conditions. The 05ms line is sterile at low temperature and fertile at high temperature. A total of 166,273,427 raw reads were obtained, 143 known miRNAs from 42 miRNA families and 104 novel miRNAs were detected. Further, six differentially expressed miRNAs (DEMs) were identified, including three known (miR168b-3p, miR397–5p, and miR408) and three novel miRNAs (Novel_116, Novel_119, and Novel_97), which might be related to anther development. Moreover, the six DEMs were validated by quantitative real-time polymerase chain reaction and 892 target genes of which were predicted. Gene Ontology analysis of target genes revealed significant enrichment in the “copper ion binding,” “oxidation-reduction process,” and “oxidoreductase activity” terms. Kyoto Encyclopedia of Genes and Genomes analysis revealed that “plant hormone signal transduction” and “other glycan degradation” were enriched. In addition, we constructed regulatory networks comprising miRNAs, target genes, and important terms/pathways and found the miR397-5p was the most linked miRNA, down-regulated under low temperature. Our findings contribute to understanding of the roles of miRNA during anther development and provide the theoretical foundation for two-line hybrid breeding of eggplant.