Somatic embryogenesis (SE) is a remarkable process of plant somatic cells developing into an embryo capable of forming a complete plant. MiRNAs play important roles in plant development by regulating expression of their target genes, but its function in SE has rarely been studied. Herein, ten conserved miRNAs with critical functions in plant development are detected by stem-loop qRT-PCR in the SE system of Valencia sweet orange. Sixteen unigenes from citrus are predicted to be targeted by six of the miRNAs. Cleavage sites on 15 of these target mRNAs are mapped by 5'RACE, of which ten are reported in this study. Transcript abundances of the 16 target unigenes are detected by qRT-PCR during SE process. Stage and tissue-specific expressions of miRNAs and their targets suggest their possible modulation on SE of citrus callus: miR156, 168 and 171 exert regulatory function during somatic embryo induction process; miR159, 164, 390 and 397 are related to globular-shaped embryo formation; miR166, 167 and 398 are required for cotyledon-shaped embryo morphogenesis; in addition, target genes of miR164, 166 and 397 are associated with SE disability of nonembryogenic callus. Exploration of miRNA-mediated modulation on SE is expected to provide new insights into plant cell totipotency, as well as how to maintain and enhance the embryogenic capacity of somatic cells.

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MicroRNAs (miRNAs) are a group of singlestranded noncoding RNAs with general size of 21-24 nucleotides, which negatively regulate gene expression posttranscriptionally by repressing gene translation or degrading targeted mRNAs. Despite the important functions of miRNAs in plants, little is known about miRNAs in citrus. Here we present a study of bioinformatics identification of microRNA precursors in citrus by comparing known plant miRNAs against 560,271 citrus ESTs. Seventy-eight ESTs from ten citrus species and three hybrids were identified as putative miRNA precursors, encoding 51 unique miRNA sequences, representing 28 miRNA families. Blastn search of the putative miRNAs against citrus unigenes and ESTs was performed to identify the target genes. As a result, 36 unigenes and 77 ESTs were identified as putative targets of 25 citrus miRNA families. The putative targets are mainly transcription factors and play important roles in development, metabolism, and stress resistance. To validate the predicted miRNAs, qRT-PCR was applied to detect the tissue-specific expression of nine putative miRNAs in Citrus sinensis cv. Valencia. The cleavage sites on five mRNAs targeted by three miRNA families were mapped by 5′RACE. This study provided information on citrus miRNA precursors, mature miRNAs, and miRNA targets and is helpful for future research of miRNA function in citrus.