Genome Assembly And Elaboration On The Flowering Regulation In Orchardgrass

Orchardgrass(Dactylis glomerata L.)is a perennial forage grasses cultivated worldwide with characteristics of high biomass yields,high carbohydrate levels,shade tolerance,and adaptability to abiotic stress,which is important for the production of foragebased meat and dairy throughout the temperate regions of the world.However,the orachradgrass genome is still largely unknown on account of high repetition rate and hybrid rate,which restrict the molecular breeding and functional genomics research of orchardgrass.Flowering time is strongly correlated with the yield and quality of forage grasses,but there were few studies on the molecular mechanisms of flowering regulation in perennial grasses.In this study,we firstly obtained 1.78 Gb high quality reference genome of diploid orchardgrass by combining use of Pac Bio single-molecule real-time(SMRT)sequencing,Illumina short-read sequencing and assisted assembly technology.In addition,we investigated the transcriptional and post-transcriptional regulation in crucial stages of flowering development and identified candidate genes of flowering regulation based on genome information.The main research results and findings are as follows:1.The construction of high-quality reference genome of orchardgrass.The 1.78 Gb orchardgrass reference genome with a contig N50 of 0.93 Mb,a scaffold N50 of 6.08 Mb,and a super-scaffold N50 of 252.52 Mb was obtained via third and next generation sequencing,Hi-C,Bio Nano and 10 X Genomics,representing the first genome assembly in a cool-season(C3)forage grass.Phylogenetic analysis reveals a common ancestor before～17.5-27.6 million years ago(Mya)between orchardgrass and three Triticeae species.One evolutionarily conserved chromosome was detected by analysing chromosome derivation in these grass species.The re-sequenced of 76 orchardgrass germplasm accessions revealed that germplasm from Northern Europe and East Asia clustered together,likely due to the exchange of plants along the ’Silk Road’ or other ancient trade routes connecting the East and West.Additionally,an online database for the orchardgrass reference genome with integrated annotations,gene blast results and transcriptomic data has been developed(https://www.orchardgrassgenome.sicau.edu.cn).This chromosome-scale genome and the online database of orchardgrass developed here will facilitate the discovery of genes controlling agronomically important traits,stimulate genetic improvement and functional genetic research,and established foundation for flowering mechanism elaboration of orchardgrass.2.RNA-seq revealed transcriptional regulation during the critical flowering periods.The successive RNA-seq during crucial flowering stages indicated that vernalization stage was the transition between vegetative growth and reproductive growth,and long-day after vernalization was the main period for flowering induction of orchardgrass.The transcriptional statistics in different stages showed 4,689 differentially expressed genes(DEGs)significantly increased in abundance,while 3,841 decreased during vernalization.Furthermore,12,969 DEGs were identified during booting stage and flowering stage,including 7,750 up-regulated and 5,219 down-regulated DEGs.Pathway analysis implied that transcripts related to circadian rhythm,photoperiod,photosynthesis,flavonoid biosynthesis,starch,and sucrose metabolism changed significantly at different stages.Comparative transcriptome analysis indicated 50 flowering-related genes were differentially expressed between early and late flowering phenotypes of orchardgrass.Of which,25flowering-related genes involved in photoperiod pathway.A combined transcriptome,quantitative genetic,and bulk segregant analysis provided insights into the genetic network regulating flowering time in orchardgrass and indicated four main candidate genes controlling this trait including three MADS-box genes and one FT-like gene.In addition,the enrichment in transcription factors(TFs)related to WRKY,NAC,AP2/EREBP,AUX/IAA,MADS-box,ABI3/VP1,b HLH,and the CCAAT family possible involved in vernalization response and floral bud development.TFs expression patterns revealed intricate temporal variations,suggesting relatively separate regulatory programs of TF modules.Further study will unlock insights into the ability of the circadian rhythm and photoperiod to regulate vernalization and flowering time in perennial grass.3.The miRNAs expression dynamic provided insights in post-transcriptional of flowering time regulation.Combine with transcriptome and miRNAs expression data in different stages,the post-transcriptional regulation of flowering was analyzed.The results revealed a total of 3,846 DEGs and 69 differentially expressed miRNAs were identified across five flowering stages.The stages statistics showed that the highest number of differentially expressed miRNAs(27)was in vernalization-after vernalization stage,accounting for 39.13% of total differentially expressed miRNAs;the highest number of DGEs in heading-booting stage(1,796),accounting for 46.70% of total DEGs.Expression pattern analysis indicated miR395,miR530,miR167,miR396,miR528,novel＿42,novel＿72,novel＿107,and novel＿123 demonstrated significant variations during vernalization stage,implied potential regulation during vernalization stage.These miRNA targeted genes were involved in phytohormones,transmembrane transport,and plant morphogenesis in response to vernalization.The expression patterns of DEGs related to plant hormones,stress responses,energy metabolism,and signal transduction changed significantly in the transition from vegetative to reproductive phases.Five hub genes,BRI1,BZR1,VRN1,VIN3,and FT,might play central roles in vernalization response and flowering time control.This work presents the high-quality genome of orchardgrass and identified important region and candidate genes of flowering trait.A comprehensive molecular expression patterns during different developmental stages at transcriptional level was described.Finally,the post-transcriptional regulation was elaborated by combining use transcriptome and miRNAs data.Acquiring a high-quality reference genome for orchardgrass is paramount to strengthening the capabilities of molecular breeding and further promoting forage grass genetic and genome-wide studies.