Analysis Of Flowering Mechanism Of Flowering Sport Chrysanthemum 'Jinba' And Elite Gene Mining Based On High-throughput Sequencing

Chrysanthemum(Chysanthemum morifolium Ramat.)is a typical short-day plant.The day length affects the flower bud formation and flowering time in chrysanthemum,but the molecular mechanism of flowering is less clear,compared with the other model plants.A chrysanthemum cultivar,designated as 'Diwenshenma'(1M),emerged inflorescence earlier one week than 'Jinba'(WT)under short day condition(SD)and could emerged flower bud under long day condition(LD).And it was a natural sport from 'Jinba' identified by the sequence-related amplified polymorphism(SRAP).We performed transcriptome sequencing between the WT and M plants under short day and long day condition at the differentiation flower bud stage,repectively,and attempt to explain the mechanism of different flowering time under different day lengths in chrysanthemum and mining elite genes.The main results and conclusions are as follows:1.The results of RNA-seq data analysis revealed the regulatory pathways of flower bud formation and flowering in M plants.Four cDNA library were constructed involving 'Jinba' mutant-LD(M-LD),'Jinba'wild type-LD(WT-LD),'Jinba' mutant-SD(M-SD)and 'Jinba' wild type-SD(WT-SD).We found many differentially expressed genes(DTGs)by cluster analysis involved photoperiod pathway,aging,ambient temperature and autonomous pathway.In SD condition,the circadian clock genes and CmFTL3 significantly upregulated inducing floral bud formation and flowering,and GA signaling pathway related genes GA20ox,GID1 and GA2ox alsoshowed significant expression level in both WT and M plants.In LD condition,GA20ox and GID1 showed upregulation,the catabolic gene GA2ox showed downregulation,resulting in CmFTL1 and CmFTL3,SOC1 upregulation and promoting floral bud formation and flowering.Also the content of endogenous GA showed higher level under long day condition in mutant compared with wild type.Furthermore,When treated with GA,the WT lines showed the similar phenotype with the M plants.These results suggested that the photoperiod and GA signalling pathways co-regulate flower bud formation and flowering time in chrysanthemum under SD,while the GA signalling pathway predominated under LD in chrysanthemum 'Jinba'.2.Cloning and function analysis of CmEMFs Gene.Besides AP1,SVP,SPL,FRI and genes related to photoperiod pathway and GA pathway,the expression levels of EMFs were changed based on the analysis of DTGs and its function was less known in chrysanthemum.Open reading frame(ORF)sequence of 2 EMF genes in 'Jinba' were confirmed by PCR.The ORF of CmEMF2.1 and CmEMF2.2 encoded 627 and 616 amino acid residues,repectively.CmEMF2.1 and CmEMF2.1 share a similarity of 58.65%,and both contained VEFS-box conserved domains.Protein sequence analysis and phylogenetic analysis showed that CmEMF2.1 and CmEMF2.2 protein had highest similarity and closer relationship.Subcellular localization results demonstrated that CmEMF2.1 localized in the nucleus.The results of real time PCR indicated that the expression of CmEMF genes were tissue-specific,and the expression level in leaf were the highest.During vegetative stage,expression level of CmEMFs increased as plant grew,and reached the peak before flower bud differentiation.After flower bud formation,the expression levels decreased,but still remained a certain level of expression.It can be speculated that CmEMFs acted to promote vegetative growth,inhibit reproductive growth,and involving in flower organ development in chrysanthemum.In addition,the expression of CmEMF2.1 responding to photoperiod signal,decreased during the day and accumulated in the dark,which may be due to that transcriptional activity is different under different lighting environment.3.Regeneration of stable transformants from Agrobacterium-mediater transiently transformed leaves in chrysanthemumThe construct pORE R1-2×35S:GUS,GUS as a reporter,was introduced into the leaves of chrysanthemum 'Yuuka' by Agrobacterium-mediated transient transformation,followed by sterilization and tissue culture.stably transformed plants were recovered,with the transformation efficiency of 38.9%which was much higher than traditional Agrobacterium-medidted infection method.That provided a new and efficient method for chrysanthemum transformation.