Integrated Analysis of Small RNA, Transcriptome, and Degradome Sequencing Reveals the Water-Deficit and Heat Stress Response Network in Durum Wheat. Integrated Analysis of Small RNA, Transcriptome, and Degradome Sequencing Reveals the Water-Deficit and Heat Stress Response Network in Durum Wheat

Water-deficit and heat stress negatively impact crop production. Mechanisms underlying the response of durum wheat to such stresses are not well understood. With the new durum wheat genome assembly, we conducted the first multi-omics analysis with next-generation sequencing, providing a comprehensive description of the durum wheat small RNAome (sRNAome), mRNA transcriptome, and degradome. Single and combined water-deficit and heat stress were applied to stress-tolerant and -sensitive Australian genotypes to study their response at multiple time-points during reproduction. Analysis of 120 sRNA libraries identified 523 microRNAs (miRNAs), of which 55 were novel. Differentially expressed miRNAs (DEMs) were identified that had significantly altered expression subject to stress type, genotype, and time-point. Transcriptome sequencing identified 49,436 genes, with differentially expressed genes (DEGs) linked to processes associated with hormone homeostasis, photosynthesis, and signaling. With the first durum wheat degradome report, over 100,000 transcript target sites were characterized, and new miRNA-mRNA regulatory pairs were discovered. Integrated omics analysis identified key miRNA-mRNA modules (particularly, novel pairs of miRNAs and transcription factors) with antagonistic regulatory patterns subject to different stresses. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis revealed significant roles in plant growth and stress adaptation. Our research provides novel and fundamental knowledge, at the whole-genome level, for transcriptional and post-transcriptional stress regulation in durum wheat. Overall design: Analysis of small RNA transcriptome in 120 samples (2 genotypes × 4 treatments × 5 time-points × 3 biological replicates); Analysis of mRNA transcriptome in eight libraries (2 genotypes × 4 treatments × 1 time-point × 1 pool of 3 biological replicates); Analysis of mRNA degradome in eight libraries (2 genotypes × 4 treatments × 1 time-point × 1 pool of 3 biological replicates)

水分亏缺与热胁迫会对作物产量产生负面影响，目前人们对硬粒小麦响应此类胁迫的分子机制尚不明晰。借助最新发布的硬粒小麦基因组组装结果，本研究首次利用下一代测序开展多组学分析，全面解析了硬粒小麦的小RNA组（small RNAome，sRNAome）、mRNA转录组以及降解组（degradome）。本研究针对澳大利亚的耐旱/热和敏感两种基因型小麦，施加单一及复合的水分亏缺、热胁迫，并在生殖生长阶段的多个时间点采集样本以分析其胁迫响应。通过对120个小RNA文库的分析，共鉴定出523个微小RNA（miRNA），其中55个为新型微小RNA。研究鉴定得到差异表达miRNA（DEMs），其表达量会随胁迫类型、基因型及采样时间点发生显著变化。转录组测序共鉴定出49436个基因，其中差异表达基因（DEGs）的富集通路涉及激素稳态、光合作用及信号转导等生物学过程。本研究首次报道了硬粒小麦的降解组，共鉴定出超过10万个转录本靶位点，并发现了全新的miRNA-mRNA调控配对。整合多组学分析鉴定得到关键的miRNA-mRNA调控模块，尤其是新型miRNA与转录因子的配对，其调控模式在不同胁迫下呈现拮抗效应。基因本体（Gene Ontology，GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）富集分析显示，这些调控模块在植物生长及胁迫适应过程中发挥重要作用。本研究从全基因组层面为硬粒小麦的转录水平及转录后水平胁迫调控机制提供了全新的基础性认知。实验设计：对120个样本开展小RNA转录组分析（2种基因型 × 4种处理 × 5个时间点 × 3次生物学重复）；对8个文库开展mRNA转录组分析（2种基因型 × 4种处理 × 1个时间点 × 3次生物学重复混合样本）；对8个文库开展mRNA降解组分析（2种基因型 × 4种处理 × 1个时间点 × 3次生物学重复混合样本）