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Small RNAs (sRNAs) are emerging as important regulators of biological processes in plants. To characterize the small RNA species and expression changes in ‘Anliu’ sweet orange (wild type, WT) and its red-flesh mutant (MT) with lycopene accumulation, we used high-throughput pyrosequencing to identify and quantitatively profile sRNAs. We identified 112 known miRNAs belonging to 64 families from the sweet orange. Comparative analysis revealed that 63 of the 112 known miRNAs exhibited significant expression differences between WT and MT. We also identified 12 novel miRNAs and 9 miRNA candidates from sweet orange; the 12 novel miRNAs are in line with the biogenesis characteristics including the stem-loop structure of the pre-miRNAs and existence of the miRNA*s in the sRNA libraries. Comparative profiling revealed that 10 novel miRNAs and 8 miRNA candidates are differentially expressed between WT and MT. Potential targets of these differentially expressed miRNAs included several important genes that are involved in carotenoid biosynthesis, such as geranylgeranyl pyrophosphate synthase (GGPS) and lycopene β-cyclase (LYCb). Moreover, GO and KEGG annotation revealed that high ranked miRNA-target genes are those implicated in transcription regulation, protein modification and photosynthesis. We proposed that miRNA-target genes are the hot-spots for generating sRNAs. This study provides the first large scale cloning and characterization of citrus miRNAs, which also lays a foundation for unraveling the mechanism of lycopene accumulation in the sweet orange mutant on post-transcription level. Size fractionated small RNAs (16-30 bp) from total RNA extracts was ligated to 5' and 3' adapters, and reverse transcribed. After PCR amplification the sample was subjected to Solexa sequencing. The resultant 35nt sequence data were filtered according to base quality value. The remained sequences were used to trim 5' and 3' adaptors. The clean tags were used for further analysis.

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