Small RNA SBS signatures identified from maize using Illumina's SBS technology

Most endogenous siRNAs in Arabidopsis are dependent on RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) for their biogenesis. Recent work has demonstrated that the maize MEDIATOR OF PARAMUTATION1 (mop1) gene is a predicted ortholog of the Arabidopsis RDR2 gene. The mop1 gene is required for establishment of paramutation and maintenance of transcriptional silencing of transposons and transgenes, suggesting the potential involvement of small RNAs. We analyzed small RNAs in wildtype maize and in the isogenic mop1-1 loss-of-function mutant using Illumina’s sequencing-by-synthesis (SBS) technology, which allowed us to characterize the complement of maize small RNAs to considerable depth. Similar to rdr2 in Arabidopsis, in mop1-1, the 24 nt (nucleotide) endogenous heterochromatic short-interfering siRNAs were dramatically reduced resulting in an enrichment of miRNAs and trans-acting siRNAs (ta-siRNAs). In contrast to the Arabidopsis rdr2 mutant, the mop1-1 plants retained a highly abundant heterochromatic ~22 nt class of small RNAs. These data suggest that maize, unlike Arabidopsis, has a second mechanism for heterochromatic siRNA production. The enrichment of miRNAs and loss of 24 nt heterochromatic siRNAs in mop1-1 should be advantageous for miRNA discovery as the maize genome becomes more fully sequenced. This library was derived from maize variety K55 wild-type and mop1 mutant. Immature ears were harvested from plants grown outdoors in Tuscon, AZ. The material was harvested from plants 68 days after germination, with the floral tissue including young tassels 2-3 cm long. Total RNA was isolated using a standard TRIZOL-based method and the small RNA libraries were generated as described by Lu et al. (Methods 2007, 43:110), followed by sequencing with Illumina's SBS method. The adapter sequences were removed, and the abundance of each distinct small RNA was determined. Raw data are unavailable from Illumina

拟南芥（Arabidopsis）中的大多数内源小干扰RNA（endogenous siRNA）的生物发生依赖于RNA依赖的RNA聚合酶2（RNA-DEPENDENT RNA POLYMERASE 2, RDR2）。近期研究证实，玉米的副突变介导因子1（MEDIATOR OF PARAMUTATION1, mop1）基因是拟南芥RDR2基因的预测直向同源基因。mop1基因对于副突变的建立以及转座子和转基因的转录沉默维持不可或缺，这提示小RNA可能参与了上述生物学过程。我们利用Illumina边合成边测序（sequencing-by-synthesis, SBS）技术，分析了野生型玉米以及同基因背景的mop1-1功能缺失突变体中的小RNA，得以在较高测序深度下表征玉米小RNA的整体组成。与拟南芥rdr2突变体类似，mop1-1突变体中的24 nt（核苷酸）内源异染色质小干扰RNA显著减少，进而导致microRNA（miRNA）和反式作用小干扰RNA（trans-acting siRNA, ta-siRNA）的富集。与拟南芥rdr2突变体不同的是，mop1-1植株仍保留了一类丰度极高的、长度约22 nt的异染色质小RNA。上述数据表明，与拟南芥不同，玉米存在第二种异染色质小RNA的生成机制。随着玉米基因组测序的逐步完善，mop1-1突变体中miRNA的富集以及24 nt异染色质小RNA的缺失，将为miRNA的发现提供便利。本测序文库源自玉米品种K55的野生型和mop1突变体材料。实验材料采自美国亚利桑那州图森市户外种植的植株，取材时为雌穗未成熟阶段，取样时间为播种后68天，采集的花组织包含长度2~3 cm的幼雄穗。总RNA的提取采用基于TRIZOL的标准方法，小RNA文库的构建参照Lu等人发表于《Methods》2007年第43卷第110页的方案，随后采用Illumina的SBS技术进行测序。测序数据首先去除接头序列，进而统计每一种独特小RNA的表达丰度。原始测序数据暂无法从Illumina获取。