Table_6_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.

冷驯化与营养/生殖生长转换是冬小麦抵御冬季低温、并于次年成功结实的两类关键进化适应机制。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核心调控作用。然而，冬小麦冷驯化及营养/生殖生长转换进程中，miRNA的调控机制目前仍不甚明晰。

本研究针对大田种植的冬小麦，分别采集三叶期、冬季休眠期、春季返青期及拔节期的叶片构建了4个sRNA文库，通过测序分析以鉴定已知miRNA与新型miRNA，并解析其在冬小麦生长发育中的潜在功能。本研究采用高通量测序技术检测miRNA的表达水平。

本次分析共鉴定得到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的表达在冬季休眠期显著受抑制，另有7个已知miRNA与8个新型miRNA的表达在该阶段被显著诱导；3个已知miRNA与3个新型miRNA在春季返青期显著上调；6个已知miRNA在春季返青期被诱导，并在拔节期达到表达峰值；另有20个已知miRNA与10个新型miRNA在拔节期显著上调。

本研究通过实时荧光定量PCR（quantitative real-time polymerase chain reaction, qRT-PCR）验证了部分代表性差异表达miRNA的表达模式。同时，对已知miRNA与新型miRNA的潜在靶基因进行了预测。此外，本研究借助RNA连接酶介导的5' cDNA末端快速扩增（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）特异性miRNA以及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。本研究为深入解析大田种植冬小麦冷驯化及营养/生殖生长转换过程中的miRNA调控机制提供了重要参考依据。