Multi-Omic Analysis On Shoot Regeneration And Genetic Transformation Efficiency Improvement In Wheat

Improving the main traits of wheat(Triticum aestivum)accurately by genetic engineering is an important approach of biotech-breeding and genetic transformation is the major foundation of genetic engineering.It is crucial to identify some key regulatory factors related to shoot regeneration in wheat tissure culture,apply them in the genetic transformation with an improved efficiency,and further explore their molecular mechanism.In this study,the immature embryos at 14 days after anthesis collected from a wheat cultivar Fielder were used as materials,cultured on callus induction medium,and then sampled at 0 day after inoculation(DAI0),DAI3,DAI6,DAI9,and DAI12 for RNA-seq,ATAC-seq(Assay for Transposase Accessible Chromatin with high-throughout sequencing)and CUT&Tag(Cleavage Under Targets and Tagmentation).Based on the expression patterns of differentially expression genes(DEGs)in RNA-seq and the chromatin accessibility of differentially accessible peaks(DAPs)in ATAC-seq and CUT&Tag at different tissue culture steps,all the potential DEGs and DAPs were grouped into C1-C6 clusters and K1-K6 clusters,respectively.Gene ontology(GO)analysis suggested that C1,K2 and K4 had high gene expression level and chromatin accessibility at DAI0,and significantly decreased during tissue culture.Osmotic stress response and fatty acid biosynthetic process were enriched with the ongoing of tissue culture.Gene transcription levels in C2,C3 and C4 and chromatin accessibility in K3 and K5 were low at the early stage of tissue culture,and gradually increased with the processing of tissue culture.The expressed genes were associated with several biological processes including response to auxin,regulation of cell population proliferation,and shoot apical meristem(SAM)development.The DEGs in C5 and the DAPs in K6 showed the highest expression levels and chromatin accessibility at DAI3.Several biological processes such as cell fate specification and auxin transport were enriched at the aforementioned culture stage.The DEGs in C6 and the DAPs in K1 were low at DAI0,while they were always high after the tissues were cultured.DNA replication initiation,t RNA transport and r RNA processing were enriched during the culture course.Correlation analysis of multi-omics data showed that gene transcription levels of each gene cluster were highly positively correlated with chromatin accessibility,negatively correlated with H3K27me3 at C2,C3,C4,and C6,positively correlated with H3K4me3 at C1 and C3,and H3K27 ac at C5.Therefore,the dynamics of gene transcription level during wheat shoot regeneration might be mainly caused by chromatin accessibility changing,and is involved in H3K27me3,H3K4me3 and H3K27ac modification.Based on the comprehensive analysis of the multi-omics data,twenty-six candidate genes encoding the corresponding regulatory factors which are potentially involved in wheat shoot regeneration were selected for functional analysis.The transformation results using Fielder showed that transformation efficiency of the expression vectors containing ten genes was higher than that of the control vector.Of them,Ta DOF3.4 gene displayed the highest transformation efficiency,Ta DOF5.6 and Ta SCR genes gave the similar results to GRF4-GIF1,which all showed higher transformation than Ta WOX5.Further experiments confirmed that Ta SCR,Ta DOF3.4 and Ta DOF5.6 effectively improved the transformation efficiency of Kenong199,Jimai22 and Aikang58.In situ hybridization analysis showed that Ta SCR and Ta DOF3.4 genes were highly expressed in embryonic cells and promeristem of callus specifically;Ta DOF5.6 transcripts were accumulated at a high level in the protocambium area of callus,but no signal was detected in embryogenic cells.By multi-omics data analysis,gene regulatory networks on Ta SCR,Ta DOF3.4 and Ta DOF5.6were conceived in the wheat shoot regeneration.In conclusion,the analysis results from the tissue culture of wheat immature embryos by RNA-seq,ATAC-seq and CUT&Tag multi-omics revealed the close relationship between the dynamic changing of gene transcription level and chromatin accessibility at different tissue culture periods during shoot regeneration.Functional analysis showed that the application of Ta SCR,Ta DOF3.4 and Ta DOF5.6 genes significantly improved the transformation efficiency of different genotypes in wheat.The results achieved in this study might provide important information and gene resources for improving the regeneration and transformation efficiency of different wheat genotypes,and will be helpful to elucidate the mechanism of wheat shoot regeneration.