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MicroRNAs (miRNAs) play essential roles in plant development, but the roles in the  in vitro  plant development are unknown. Leaves of ponkan plantlets derived from mature embryos at  in vitro  culture conditions were used to sequence small RNA fraction via Solexa sequencing, and the miRNAs expression was analyzed. The results showed that there were 3,065,625 unique sequences in ponkan, of which 0.79% were miRNAs. The RNA sequences with lengths of 18–25 nt derived from the library were analyzed, leading to the identification of 224 known miRNAs, of which the most abundant were miR157, miR156, and miR166. Three hundred and fifty-eight novel miRNA candidates were also identified, and the number of reads of ponkan novel miRNAs varied from 5 to 168,273. The expression of the most known miRNAs obtained was at low levels, which varied from 5 to 4,946,356. To better understand the role of miRNAs during the preservation of ponkan  in vitro  plantlet, the expression patterns of  cre-miR156a/159b/160a/166a/167a/168a/171/398b  were validated by quantitative real-time PCR (qPCR). The results showed that not only the development-associated miRNAs, e.g.,  cre-miR156 / 159 / 166/396 , expressed highly at the early preservation period in the  in vitro  ponkan plantlet leaves but also the stress-related miRNAs, e.g.,  cre-miR171  and  cre-miR398b , expressed highly at the same time. The expression levels of most tested miRNAs were found to decrease after 6 months and the amounts of these miRNAs were kept at low levels at 18 months. After analyzing the expression level of their targets during the reservation of the ponkan  in vitro  plantlet, development-associated  cre-ARF6  and stress-related  cre-CSD  modules exhibited negative correlation with  miR167  and  miR398 , respectively, indicating an involvement of the miRNAs in the  in vitro  development of ponkan and function in the conservation of ponkan germplasm.

Identification of Novel and Conserved miRNAs in Leaves of In vitro Grown Citrus reticulata “Lugan” Plantlets by Solexa Sequencing