| Fritillaria spp.are perennial bulb plants that belong to the Liliaceae family,and have important medicinal and ornamental value.In this study,the biological characteristics and the geographical parameters of seven important wild species were record and the application value of the garden was initially analyzed after the collection of wild Fritillaria resources in Xinjiang,China.Through the introduction and cultivation of 18 Fritillaria spp.induced from home and abroad,the adaptability evaluation and in vitro rapid propagation technology were studied,and six early flowering cultivars suitable for open cultivation in Beijing were screened.At the same time,in vitro rapid propagation system of five species inclued F.pallidiflora were established.In addition,through the cytological observation of nine species belonged to three subgenera(Subg.Petillium,Fritillaria and Rhinopetalum),their chromosome karyotype formula were record,the karyotype pattern were constructed,and their kinship relationship were also analyzed.Furthermore,the main parameters of pollen grains and the characteristics of pollen exine sculpture of 16 Fritillaria samples belonged to four subgenera(Subg.Petillium,Theresia,Fritillaria and Rhinopetalum)were record.This provided palynology basis for the study of system evolution and species identification.Finally,the internal mechanism of F.pallidiflora flower bud differentiation was studied by morphological observation,physiological index determination and RNA-seq technique,in order to lay the theoretical foundation for the mechanism of flower bud differentiation and the application of flowering control technology from the levels of transcription and microRNA to analyze the mechanism of gene regulation.The main results were as follows.(1)Resource collection,introduction and cultivation of wild fritillaries in Xinjiang had been conducted.The natural habitats and biological characteristics of seven wild fritillaries in Tacheng area,Xinjiang were record,and the garden application value of their wild resources was initially analyzed.The 18 species(cultivars)introduced at home and abroad were cultivated,and their adaptation was also evaluated.After evaluation analysis,six species were selected and were suitable for popularization and application in Beijing area.(2)In vitro propagation technology of Fritillaria species was researched and the rapid propagation system of five species(included F.pallidiflora)were established.Among them,the callus proliferation coefficient of F.anhuiensis was up to 3.15,and average per explant can induce 11.3 bulblets at maximum on F.thunbergii.The average number of adventitious buds inducing proliferation was 7.6 on F.verticillata,and its proliferation coefficient can reach to 1.81.Moreover,the bulblets induction efficiency of F.pallidiflora can achieve 83.33%.In addition,the seeds of F.persica could germinate on the medium with MS+30 g/L sucrose.This method could shorter nearly two years than the time of open field sowing need.(3)Genetic analysis of chromosome karyotype and ultrastructure of pollen were carried on.Karyotypes of nine species belonged to three subgenera were studied using squashing method.The results showed that eight Fritillaria species were diploid(2n=2x=24),except F.uva-vulpis,which was triploid(2n=3x-3=33).The karyotypes of F.pallidiflora,F.tortifolia and F.verticillata var.albidoflora were 2B,and the other species were 3B.In addition,the clustering analysis of karyotype resemblance-coefficient was conducted among eight Fritillaria species.In the study of pollen ultrastructure,the pollen grains of 16 species or cultivars belonged to four subgenera were observed and examined under a scanning electron microscope(SEM).The results showed that the pollen grains of all studied species were subprolate to perprolate,with bilateral symmetry,single-colporate,almost up to the poles,oblong-ellipsoidal in equatorial view,suborbicular in polar view.The surface of the pollen grains was mostly with the type of reticulate exine sculpturing.The meshes were obviously larger or slightly larger than the width of the ridge.The width of ridge was various and was consisted of single,double or multi-row particles.Then,the pollen viability of fritillaries were measured,and the results showed that the pollen vitality of F.pallidiflora was the highest,followed by F.tortifolia,F.meleagris and F.verticillata var.albidoflora.Pollen had the highest germination rate in the optimal combination of culture medium was 0.1%boronic acid+15%sucrose.In addition,it was especially benefit to keep the pollen vitality in dry conditions when the temperature was-20?.The cytology study showed that the fritillaries in the same subgenus had similar karyotype and pollen morphology,and their genetic relationship was relatively close.In addition,the pollen morphology was more regular,the pollen vitality or germination rate was relatively higher.(4)The determination of flower bud differentiation period of F.pallidiflora was studied.The flower bud differentiation will complete at 25/18?(day/night),12 h/d for about 80 d under the condition of sand.The differentiation process could be divided into six stages:undifferentiated,bracteatum differentiation,petal primordium differentiation stage,stamen primordium differentiation stage and pistil primordium differentiation stage.In this process,the content of soluble sugar and protein in flower buds increased first and then decreased,and respectively reached the maximum value at different stages of petal primordium differentiation and stamen primordia.Moreover,the starch content decreased to the lowest value in the stamen primordium differentiation period.Except for the change of gibberellin(GA)content no obvious,the other three hormones were in a low valleys at the petal primordium differentiation stage,while the relative contents of the hormones showed a dynamic change state,no obvious regularity.Therefore,transcriptions and small RNA sequencing analysis were performed using RNA-seq technique for the three developmental stages of the undifferentiated(F1),petal primordium differentiation(F2)and differentiation completion period(F3).(5)Transcriptome of F.palliddiflora different stages of flower bud differentiation was analyzed.The database of transcriptome was constructed from the three stages of flower bud differentiation.A total of 74,353 unigenes were obtained after sequencing,with an average length of 782 bp.Of these,34288(46.12%)unigenes were annotated with protein function,and 1216 unigenes were annotated to 57 transcription factor families.Through differentially expressed gene analysis,it was found that there were 3402 genes expressed in the three comparison groups,and GO and KEGG-Pathway enrichment were analyzed.There were 2645 genes differentially expressed only in petal primordium differentiation,and the genes related to sugar metabolism,plant hormones,transcription factors,histones and methyltransferases were also differentially expressed.In addition,the genes of FPA,AGL19,TFL1,ELF3,LUG,CO,SPLs,PHYC,etc.related to flower development were screened out.They were significantly different in the process of flower bud differentiation,indicating that these genes were involved in flower bud differentiation process.But its specific regulatory function has yet to be the next step to verify the test.(6)Expression of microRNA in the process of flower bud differentiation of F.pallidiflora was analyzed.The results of sequencing of microRNA were processed and compared with miRBase database.98 known miRNAs belonged to 77 miRNA families were found and 195 novel miRNAs were predicted in three samples.In addition,285 miRNAs were predicted for target genes,and 2816 target genes were predicted,simultaneously,4288 target gene loci were predicted.The results of GO enrichment analysis showed that they were mainly enriched in metabolic process,cellular process,catalytic activity,binding,cell part,cell and so on.The results of KEGG-Pathway enrichment analysis showed that they were mainly enriched in carbon metabolism,biosynthesis of amino acids,protein processing in endoplasmic reticulum,and so on.Analysis of differentially expressed miRNAs revealed that there were 36 known-miRNAs and 61 novel-miRNAs differentially expressed only in petal primordium differentiation.Of these,miR156,miR157,miR165,miRl67,miRl69 and miR319 may play a role in regulating the flower bud differentiation of F.pallidiflora.But their actual functions need to be verified through experiments.Through the analysis of transcriptome and microRNA,it was found that 11 differentially expressed microRNAs targeted negative control of 19 mRNAs and they can be considered as candidate gene for further research.In summary,this study provides theoretical and practical basis for the garden application of Fritillaria spp.,in vitro propagation,system evolution and molecular mechanism of flower development.At the same time,it also lays the foundation for the development and utilization of Fritillaria L.resources in China. |
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