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

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... 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.