Identification and temporal expression analysis of conserved and novel microRNAs in the leaves of winter wheat grown in the field

Abstract

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 (miRNAs) 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 three-leaf stage, winter dormancy stage, spring greenup stage, and jointing stages 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 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 in spring greenup stage; six known miRNAs were induced in spring greenup stage and reached the highest expression level in jointing stage; twenty known miRNAs and ten novel miRNAs were significantly induced at the jointing stage. Expression of a number of representative differentially expressed miRNAs was verified using 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 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 the regulatory mechanism of miRNAs in cold acclimation and vegetative/reproductive transition of winter wheat grown in the field. visional