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

Abstract

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.

Small RNAs and their targets are associated with the transgenerational effects of water-deficit stress in durum wheat

“…Flag leaf RNA and developing grain RNA samples from DBA Artemis were used for small RNA, transcriptome and degradome sequencing analysis (Supplementary Table S2). The sRNA libraries were prepared using the NEBNext Multiplex Small RNA Library Prep Kit and multiplex primers, as previously described 26,28,29 . sRNA-seq was performed on Illumina HiSeq 2500 at LC-Bio in Hangzhou, China.…”

“…Original datasets of sRNA-seq, transcriptome-seq, and degradome-seq generated in the current study were submitted to NCBI GEO database (accession number GSE162008). Conserved and novel durum wheat miRNAs were identified using the ACGT101-miR program as previously described 26,28,29 . All miRNAs were categorised into five groups (G1-5), where G1 to G4 represent different categories of conserved miRNAs, while G5 represents novel miRNAs.…”

“…The transcriptome libraries ( Supplementary Table S2) were constructed using the Illumina mRNA-Seq sample preparation kit, and sequenced on Illumina NovaSeq 6000 at LC-Bio as previously described 28 . Bioinformatics analysis of transcriptome-seq was performed as previously described 28 . For details, see Methods S1.…”

“…The degradome libraries ( Supplementary Table S2) were prepared and sequenced on Illumina HiSeq 2500 at LC-Bio as previously described 28 . Bioinformatics analysis of degradome-seq was performed using the CleaveLand package and the ACGT101-DEG program as previously described 28 . Degradome-seq signatures were analysed to determine mRNA targets that were post-transcriptionally regulated by durum miRNAs.…”

“…of T. turgidum DEGs was performed regarding their biological processes, cellular components and molecular functions 28,29 . KEGG pathway [30][31][32] enrichment analysis was conducted to determine the functional pathways that DEGs were associated with.…”