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Cold acclimation and vegetative/reproductive transition are two important evolutionary adaptive mechanisms for winter wheat surviving the freezing temperature in winter and successful seeds setting in the next year. MicroRNA (miRNA) is a class of regulatory small RNAs (sRNAs), which plays critical roles in the growth and development of plants. However, the regulation mechanism of miRNAs during cold acclimation and vegetative/reproductive transition of winter wheat is not much understood. In this study, four sRNA libraries from leaves of winter wheat grown in the field at the three-leaf stage, winter dormancy stage, spring green-up stage, and jointing stage were analyzed to identify known and novel miRNAs and to understand their potential roles in the growth and development of winter wheat. We examined miRNA expression using a high-throughput sequencing technique. A total of 373 known, 55 novel, and 27 putative novel miRNAs were identified. Ninety-one miRNAs were found to be differentially expressed at the four stages. Among them, the expression of six known and eight novel miRNAs was significantly suppressed at the winter dormancy stage, whereas the expression levels of seven known and eight novel miRNAs were induced at this stage; three known miRNAs and three novel miRNAs were significantly induced at the spring green-up stage; six known miRNAs were induced at the spring green-up stage and reached the highest expression level at the jointing stage; and 20 known miRNAs and 10 novel miRNAs were significantly induced at the jointing stage. Expression of a number of representative differentially expressed miRNAs was verified using quantitative real-time polymerase chain reaction (qRT-PCR). Potential target genes for known and novel miRNAs were predicted. Moreover, six novel target genes for four  Pooideae  species-specific miRNAs and two novel miRNAs were verified using the RNA ligase-mediated 5′-rapid amplification of cDNA ends (RLM-5’RACE) technique. These results indicate that miRNAs are key non-coding regulatory factors modulating the growth and development of wheat. Our study provides valuable information for in-depth understanding of the regulatory mechanism of miRNAs in cold acclimation and vegetative/reproductive transition of winter wheat grown in the field.

Identification and Temporal Expression Analysis of Conserved and Novel MicroRNAs in the Leaves of Winter Wheat Grown in the Field