Table_4_Identification of MicroRNAs and Their Targets That Respond to Powdery Mildew Infection in Cucumber by Small RNA and Degradome Sequencing.XLSX

Powdery mildew (PM) is a prevalent disease known to limit cucumber production worldwide. MicroRNAs (miRNAs) are single-stranded molecules that regulate host defense responses through posttranscriptional gene regulation. However, which specific miRNAs are involved and how they regulate cucumber PM resistance remain elusive. A PM-resistant single-segment substitution line, SSSL508-28, was developed previously using marker-assisted backcrossing of the PM-susceptible cucumber inbred D8 line. In this study, we applied small RNA and degradome sequencing to identify PM-responsive miRNAs and their target genes in the D8 and SSSL508-28 lines. The deep sequencing resulted in the identification of 156 known and 147 novel miRNAs. Among them, 32 and six differentially expressed miRNAs (DEMs) were detected in D8 and SSSL508-28, respectively. The positive correlation between DEMs measured by small RNA sequencing and stem-loop quantitative real-time reverse transcription–polymerase chain reaction confirmed the accuracy of the observed miRNA abundances. The 32 DEMs identified in the PM-susceptible D8 were all upregulated, whereas four of the six DEMs identified in the PM-resistant SSSL508-28 were downregulated. Using in silico and degradome sequencing approaches, 517 and 20 target genes were predicted for the D8 and SSSL508-28 DEMs, respectively. Comparison of the DEM expression profiles with the corresponding mRNA expression profiles obtained in a previous study with the same experimental design identified 60 and three target genes in D8 and SSSL508-28, respectively, which exhibited inverse expression patterns with their respective miRNAs. In particular, five DEMs were located in the substituted segment that contained two upregulated DEMs, Csa-miR172c-3p and Csa-miR395a-3p, in D8 and two downregulated DEMs, Csa-miR395d-3p and Csa-miR398b-3p, in SSSL508-28. One gene encoding L-aspartate oxidase, which was targeted by Csa-miR162a, was also located on the same segment and was specifically downregulated in PM-inoculated D8 leaves. Our results will facilitate the future use of miRNAs in breeding cucumber varieties with enhanced resistance to PM.

白粉病（PM）是一种全球范围内限制黄瓜生产的常见病害。微小RNA（miRNAs）是一类通过转录后基因调控调节宿主防御反应的单链RNA分子。然而，具体哪些miRNAs参与其中，以及它们如何调控黄瓜对白粉病的抗性，目前仍不明确。此前，研究人员通过对感白粉病的黄瓜自交系D8进行标记辅助回交，成功培育出抗白粉病单片段替换系SSSL508-28。本研究采用小RNA和降解组测序技术，在D8与SSSL508-28两个品系中鉴定响应白粉病的miRNAs及其靶基因。深度测序共鉴定得到156个已知miRNAs和147个新型miRNAs。其中，在D8和SSSL508-28中分别检测到32个和6个差异表达miRNAs（DEMs）。通过茎环定量实时反转录聚合酶链式反应对DEMs的表达量进行验证，结果与小RNA测序结果呈显著正相关，证实了所观测到的miRNA表达丰度的准确性。在感病品系D8中鉴定得到的32个DEMs均表现为上调表达，而在抗病品系SSSL508-28中鉴定得到的6个DEMs中有4个表现为下调表达。本研究通过计算机预测（in silico）与降解组测序技术，分别在D8和SSSL508-28的DEMs中预测得到517个和20个靶基因。将DEMs的表达谱与此前采用相同实验设计的研究中获得的对应mRNA表达谱进行比对，分别在D8和SSSL508-28中鉴定得到60个和3个靶基因，这些靶基因的表达模式与对应miRNAs呈负相关。尤为值得注意的是，有5个DEMs位于替换片段区域，其中在D8中存在2个上调表达的DEMs：Csa-miR172c-3p与Csa-miR395a-3p；在SSSL508-28中存在2个下调表达的DEMs：Csa-miR395d-3p与Csa-miR398b-3p。由Csa-miR162a靶向的、编码L-天冬氨酸氧化酶的基因同样位于该替换片段区域，且在接种白粉病的D8叶片中呈现特异性下调表达。本研究结果将为后续利用miRNAs培育抗白粉病能力提升的黄瓜品种提供重要参考。