High-throughput sequencing of small RNAs in Hordeum vulgare. Hordeum vulgare

Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from different Hordeum vulgare tissues (leaves, inflorescence and leaves inoculated with Blumeria). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the maize genome under study. Overall design: Small RNA libraries were derived from leaves, inflorescence and leaves inoculated with Blumeria of Hordeum vulgare. Each tissue represented a mixture of developmental stages. Total RNA was isolated using the Plant RNA Purification Reagent (Invitrogen) and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA libraries were sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank Roger Wise for providing the plant material and Kan Nobuta and Gayathri Mahalingam for assistance with the computational methods..

小RNA（Small RNAs，21~24 nt）是基因表达的关键调控因子，可介导包括几乎所有植物在内的多种真核生物中的转录及转录后基因沉默机制。微RNA（MicroRNAs，miRNAs）与小干扰RNA（short interfering RNAs，siRNAs）是两类主要的小RNA，二者均被证实对植物生长发育、胁迫响应及病原抗性具有重要调控作用。本研究采用深度测序技术（边合成边测序，Sequencing-By-Synthesis，简称SBS），对不同大麦（Hordeum vulgare）组织中的小RNA进行测序以获取序列资源，所分析的组织包括叶片、花序以及接种布氏白粉菌（Blumeria）的叶片。所得数据集的测序深度较高，使得我们能够详细分析该物种中小RNA的关键特征，包括其长度分布、组织特异性调控模式以及不同器官间的序列保守性。本研究还构建了相关数据库资源，并搭建了专用网站（http://smallrna.udel.edu/），配套提供计算工具，以便其他研究者鉴定参与特定调控通路的新型微RNA或小干扰RNA，验证这些序列在其他植物物种中的保守程度，以及将小RNA定位到所研究的玉米基因组的基因区域或更大基因组区段上。实验整体设计：小RNA文库来源于大麦的叶片、花序以及接种布氏白粉菌的叶片，每个组织样本均包含多个发育阶段的材料。采用植物RNA纯化试剂（Invitrogen）提取总RNA，随后送至加利福尼亚州海沃德市的Illumina公司（http://www.illumina.com），参照Lu等人2007年发表的方法并略作修改，构建小RNA文库。Illumina公司采用边合成边测序（SBS）技术对小RNA文库进行测序。研究人员设计PERL脚本以去除接头序列，并统计每一种独特小RNA的表达丰度。感谢Roger Wise提供植物材料，以及Kan Nobuta与Gayathri Mahalingam在计算方法方面提供的协助。