NGS Transcriptome of Small RNAs in Rice Male Gametophyte Development

Small RNAs, designated as miRNAs and siRNA, are classes of 19-25 nucleotide RNAs, and are considered to have important roles for an epigenetic regulation of gene expression in various scenes. In this study, we performed identification and functional analysis of small RNAs associated with male gametophyte differentiation and maturation in rice. In order to acquire comprehensive data of small RNAs in the developing anther, four small RNA-cDNA libraries were constructed from rice anther according to developmental stages, i.e. meiosis/tetrad, unicellular microspore, bicellular pollen and tricellular pollen. These libraries were sequenced by next generation sequencing technology, and their RNA sequences were subjected to BLAST analysis against the rice genome and non-coding RNAs. Consequently, a majority of known rice small RNAs and novel small RNA candidates were detected in these libraries. To identify novel miRNA, we predicted new miRNAs and their target genes. To confirm the functional miRNAs regulating target gene expression in the male gametophyte, we adopted an inverse correlation analysis between predicted expression profile of new miRNA candidates and expression datasets of these target genes from LM-44k microarray performed on developing pollen and tapetum cells. Consequently, 19 reported miRNAs and 162 novel miRNA candidates were obtained as functional miRNA in rice male gametophyte development. Taken together, we obtained many kind of small RNA information on male gametophyte development in rice, by a combination of NGS transcriptome and bioinformatics analysis. These results indicate that small RNA-mediated gene regulation operates an accurate male gamete development.