Table_2_Identification and Temporal Expression Analysis of Conserved and Novel MicroRNAs in the Leaves of Winter Wheat Grown in the Field.xlsx

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.

低温驯化与营养/生殖生长转换是冬小麦适应冬季低温冻害、并于次年顺利结实的两类重要进化适应性机制。微小RNA（microRNA，miRNA）是一类调控型小RNA（small RNAs，sRNAs），在植物生长发育过程中发挥关键调控作用。然而，冬小麦低温驯化及营养/生殖生长转换过程中miRNA的调控机制尚未得到充分阐释。
本研究针对田间种植的冬小麦，选取三叶期、冬季休眠期、春季返青期及拔节期的叶片构建了4个小RNA文库，通过高通量测序技术鉴定已知miRNA与新型miRNA，并解析其在冬小麦生长发育中的潜在功能。本研究共鉴定得到373个已知miRNA、55个新型miRNA以及27个推定新型miRNA；在四个发育阶段中共筛选得到91个差异表达miRNA。其中，6个已知miRNA与8个新型miRNA在冬季休眠期表达显著下调，另有7个已知miRNA与8个新型miRNA在该阶段表达显著上调；3个已知miRNA与3个新型miRNA在春季返青期表达显著上调；6个已知miRNA在春季返青期表达上调，并在拔节期达到表达峰值；20个已知miRNA与10个新型miRNA在拔节期表达显著上调。
研究采用实时荧光定量聚合酶链式反应（quantitative real-time polymerase chain reaction, qRT-PCR）对部分代表性差异表达miRNA的表达水平进行了验证；同时预测了已知miRNA与新型miRNA的潜在靶基因。此外，通过RNA连接酶介导的cDNA末端快速扩增5′端（RNA ligase-mediated 5′-rapid amplification of cDNA ends, RLM-5’RACE）技术，验证了4个早熟禾亚科（Pooideae）物种特异性miRNA及2个新型miRNA的6个新型靶基因。
上述结果表明，miRNA是调控小麦生长发育的关键非编码调控因子。本研究为深入解析田间种植冬小麦低温驯化及营养/生殖生长转换过程中的miRNA调控机制提供了重要参考依据。