Small RNA sequencing analysis of pepper

To facilitate the functional annotation of the pepper genome, analysis of miRNAs was performed for the sequenced data from five small RNA libraries described above, representing five different tissues. Starting with a set of 5,436 plant mature miRNA sequences available in miRBase, we annotated with high confidence 176 pepper miRNAs from 64 families, of which 30 families are computationally predicted to target TFs, suggesting important roles of these miRNA families in post-transcriptional gene regulation and transcription networks consistent with previous findings. Overall design: To identify genomic regions generating small RNAs, we applied previously described analytical strategies to five small RNA libraries from different tissues. Developing roots, stems and mature leaves were collected from plants grown in soil in a green house at 22 °C with a 16 hr light cycle and harvested from plants at full-bloom stage. Mature plants were harvested for fully open flowers. Additional flowers were allowed to self-pollinate and fruit was harvested in the breaker stage (thirty days after pollination when the fruit was turning red). Total RNA for different tissues was isolated from the frozen root samples by using the Trizol Reagent (Invitrogen) according to manufacturer's instructions, and libraries were constructed using the Small RNA Sample Prep Kit (Illumina, San Diego, CA) as previously described, then sequenced on an Illumina HiSeq2000 system.

为推进辣椒基因组的功能注释研究，我们针对上述5个涵盖不同组织的小RNA测序文库数据，开展了微RNA(miRNA)的分析工作。我们以miRBase数据库中收录的5436条植物成熟微RNA序列作为初始数据集，经注释最终获得了置信度较高的176条辣椒微RNA，分属64个家族；其中30个家族经计算预测可靶向转录因子(Transcription Factor, TFs)，这表明这些微RNA家族在转录后基因调控与转录网络中发挥着重要作用，与既往研究结论相符。实验整体设计如下：为鉴定产生小RNA(small RNA)的基因组区域，我们将既往报道的分析策略应用于5个源自不同组织的小RNA文库。我们从温室中22℃、16小时光照周期的土壤培养植株上，采集了处于盛花期的发育根、茎和成熟叶片；同时采集成熟植株的完全开放花朵。另有部分花朵经自花授粉后，于转色期（授粉后30天、果实刚转红时）采收果实。我们采用Trizol试剂(Invitrogen)按照产品说明书，从各冰冻组织样本中提取总RNA，随后使用小RNA样本制备试剂盒(Illumina, 加利福尼亚州圣地亚哥)构建测序文库，操作流程与既往报道一致，最终在Illumina HiSeq2000测序系统上完成测序。