Multi-omics Assisted Identification Of The Key Drought Tolerant Mechanisms In Gossypium Stocksii

Cotton(Gossypium spp.)is an importantly economic fiber and oil crop,and provides natural textile materials.Upland cotton is the main cultivated species.Although the cultivated species have high fiber length and yield due to long-time artificial breeding,the germplasm resources of upland cotton are relatively single,which results in its weak ability to adapt to environmental stresses.Gossypium stocksii is one of the wild cotton species,with high resistance to diseases,insects,and drought,and is therefore a valuable genetic resource for breeding and improvement of cultivated accessions.Nevertheless,the molecular mechanisms of G.stocksii response to environmental stresses are rarely known.This work performed a systematic analysis of the key genes and pathways involved in G.stocksii drought tolerance by integrating multi-omics data and the regulatory networks.The main results are as follows:1.We generated a chromosomal-level genome assembly of G.stocksii.The resulting assembly was about 1.45 Gb in length,with 13 established chromosomes accounting for 99.2%.The genome annotation was performed based on de novo and evidence-based methods,resulting in the identification of 46,224 coding genes that were largely conserved across plant species.In addition,repetitive sequences accounted for 65.02% of the genome in length,which was one of the main factors affecting the genome size during the evolution scenario.2.Comparative genomics revealed the large-scaled genomic synteny between G.stocksii and other Gossypium species,indicating relatively conserved genome structures and gene content within the genus Gossypium.Phylogenetic analyses based on the single-copy genes showed that consistent with previous knowledge,G.stocksii is closely related to the A and G genomes in the clades of African and Asia clade and Australian clade.We also found the expansion and contraction of a series of gene families in G.stocksii during speciation,which was closely associated with the functions such as abscisic acid-activated signaling pathway and cell structure stability and possibly contributed to environmental adaptation.3.We comparatively analyzed the transcriptomes of G.stocksii seedling leaves under drought stress condition.4,484 genes were differentially expressed(DEGs)in the PEG treated plants.Functional analyses showed these DEGs were enriched in the biological processes such as systemic acquired resistance,response to water,and photosynthesis.In addition,we also found that the expression modulation of a series of transcription factors within ERF,MYB,b HLH,and NAC families were involved in G.stocksii response to drought stress.4.We performed a genome-wide identification of transcription factor binding sites(TFBSs)within promoter regions of G.stocksii coding genes.These regulations were further filtered based on the co-expression networks constructed using the expression profiles of several G.stocksii tissues.Finally,transcription factor enrichment analyses revealed that 69 TFs played crucial roles in G.stocksii drought tolerant mechanisms,including the well-known genes like ABI5 and ABF2.5.We identified 285 miRNAs expressed in G.stocksii seedling leaves through small RNA sequencing,with 239 conserved across plant species.Comparative analyses showed that 20 miRNA genes were significantly down-regulated under PEG condition.While checking the miRNA targets and their expression variation after PEG treatment,we found that the miR164-NAC1 module might be one of the key regulatory components involved in the G.stocksii response to drought stress.In summary,based on the high-quality genome assembly,this project systematically identified the key genes and regulatory pathways involved in G.stocksii drought stress response through the integrative analyses of multi-omics data.This project provided data basis and new evidence for the phylogeny of cotton species,deepened our understanding of the molecular mechanism of wild cotton’s stress resistance,and promoted and guided the application of wild cotton in cultivated cotton breeding.