| Dendrobium catenatum is one of the valuable Chinese traditional medicine,which also has great ornamental value.Tissue culture has been proven to be an effective way to utilize and protect this endangered medicinal plant species.This method needs exogenous addition of plant hormones,which are commonly cytokinin and auxin,and cytokinin alone in large-scale industrial production.However,this raising process often produces different proportions of abnormal seedlings,seriously affecting the efficiency of large-scale production.Meanwhile,the large-scale production is likely to cause diseases resulting from hormones application.Therefore,it is necessary to study the morphological distortion(such as overwhelming branching,rootless and dwarfism)induced by cytokinins under tissue culture conditions as a kind of pathological changes.In this thesis,we carried out comparison analysis between D.catenatum and five other confusion Dendrobium species,including morphological description and phylogenetic tree construction.Through these analysis,we identified a new Dendrobium species and choose D.catenatum collected from Guangnan,Yunnan province as our research object.By treating the tissue culture seedlings cytokinins at three different concentrations and analyzing the morphological changes,the hormone-responsive concentration range was determined.Afterwards,we established a score index for the morphological changes and defined the disease as exogenous hormone-induced tissue culture seedlings witches' broom.Finally,the transcriptional analysis was carried out by extracting the total RNA from each treatment and using the strategy of splitting the phenotypic traits of the disease,and the molecular basis of the disease was preliminarily revealed by the identification of the related orthologous genes.The main results and conclusions are as follows:1.Identification of a new species.In the course of comparative analysis of D.catenatum,we found a new type of Dendrobium.By sampling 112 species of Dendrobium and plants from four close genus which were used as outgroups,a phylogenetic tree was constructed.The results suggested that the new species is clustered together with D.catenatum and D.dendrobium,gathering in the sect.Dendrobium.There was a significant difference between this species and other species in pollen and flower shape.Based on the results of phylogenetic analysis and morphological analysis,the new species was identified as a new species of Dendrobium,named as Dendrobium Bannaense YQ Tian&YB Huang.2.Determination of the hormone-responsive concentration range of tissue culture seedlings.By testing effects of hormones at the initial,the refined and the reference concentrations,it was found that the seedlings were differently sensitive to hormones in the ranges of 2 mg·L-1 and 20 mg·L-1,respectively.Interestingly,variations of concentration within one range have similar effects on symptoms,while hormones at different concentration ranges influenced phenotypes at significant different levels.The results showed that of hormone treatments at low concentrations impacting the bud growth,root growth and root formation,and plant height and other phenotypic traits need relatively long time periods,whereas high concentrations hormones treatments induced severer symptoms within short time.3.Classification of exogenous hormone-induced witches' broom.In order to further study the phenotypic characteristics of hormone-induced witches' broom,adopting the average number of buds as the main symptom definition index,the symptoms were classified into four grades,from grade 0 which represents normal phenotype to grade 3 that means being dead within short time.In detail,grade 1 is the almost normal type,the corresponding concentration of causal hormones was 1.5 mg ·L-1,or at a lower concentration which requires 6-month incubation time;grade 2 is the multi-buds and rootless type(M-S),the corresponding concentration of causal hormones was 19.5 mg·L-1,or at a lower concentration which requires 6-month incubation time;the grade 3 is the multi-buds and-branches with less root type(H-S),the corresponding initial concentration of hormone was 19.5mg·L-1,and was cultured for more than 3 months.4.RNA extraction and transcriptomes De novo assembly.The seedlings at grades 1,2 and 3 showed remarkable divergence in height,bud and root develpment.Aiming to reveal the underlying molecular mechanism,the transcriptomes analyses were performed.The total RNA was extracted from seedlings showing symptoms at three grades respectively,followed by the next generation sequencing(NGS),library construction and De novo assembly.The contigs and unigenes datasets were then obtained.In addition,we perform the normalization and calculated the relatively expression quantity,which set a foundation for further analysis.5.Transcriptome analyses.After the assembly,we 'split' the morphological features and then combined certain strategies to analyze the transcriptome.The results suggested among the differentially expressed genes 1996 genes were correlated with branching,119 genes were involved in rootless,166 genes related to dwarf and 6 genes was simultaneously involved in branching,rootless and dwarf.These 6 genes are c71492-g1_i1|m.34282,c57389_g1_i1|m.272938,c60692_g1_i1|m.185242,c70260_g1_i2|m.84444,c62908_g4_i1|m.229979 and c72344_g1_i2|m.135613.The fore three genes might act as signal molecules and participate in metabolization of the ubiquitin proteasome system to sustain the morphological changes.The following three genes presents lectin activity and may be involved in the synthesis of cell wall to sustain the responses towards cytokinin treatment.6.Identification of orthologous genes.In order to overcome the disadvantages of the 'split' strateage and the strict threshold setting,it is feasible to compare our transcriptome results with published genomic data to identify the cytokinin orthologous and symptoms correlated orthologous genes.Therefore,eighteen NJ trees with reasonable topological structure and high support ability were constructed.90 genes related to the metabolism of cytokinin in D.catenatum were identified,which is 53.25%of templates.Among them,there are 51 genomic data accounting for 56.7%,while the number of unigene transcriptome data is 31 accounting for 43.3%.These genes are involved in the synthesis,transport and transduction,and response and regulation of cytokinins.These data enable a more comprehensive analysis of the cytokinin metabolism in our samples.In the latter on identification,a total of 174 genes closely related to the target phenotypes were identified.Among them,there are 91 genomic data accounting for 52.3%,while 83 cases of unigene transcriptome data accounting for 47.7%.These genes are involved in stem and shoot branching and axillary bud releasing,root organ developing and root formatting,as well as plant growth repressing.All the above-mentioned data provides a more comprehensive base for analyzing the underlying molecular mechanism of witches' broom.7.KEGG analysis of symptoms related genes.The results of transcriptome analysis and the identification of orthologous genes were applied with KEGG pathway analysis.The results showed that among the rootless related differentially expressed genes,c55572_g1_il|m.254717,c78288_g2_i4|m.176933,c74031_g1_i1|m.55408 and c61756_g1_i2|m.89069 and their respective orthologous genes DcaAHP4,DcaAHP6,DcaA-ARR8 and DcaB-ARR11 could be annotated into the same metabolic pathways.The dwarf related differentially expressed gene c70319_g5_i1|m.98190 and its orthologous genes DcaDELLA?DcaAHP4?DcaAHP6?DcaA-ARR8 and DcaB-ARRll are able to be annotated into the same metabolic pathways.The branching reated differentially expressed gene c65683_g1_i3|m.146449 and its orthologous gene DcaABCG25 can to be annotated into the same metabolic pathway.These results suggested that the symptoms related genes are relevant in the symptoms of metabolic pathways.They also indicated our 'split'strategy and the orthologous genes identification method complement well.8.Signal flow of CK transportation.Through identification of cytokinin orthologous genes and combining their differential expression patterns in seedlings exhibiting symptoms at different levels,it was speculated that DcaENTl might mediate transportation of cytokinin iP from the sprout to the root.Moreover,DcaENT1 is likely to crosstalk with DcaABCG14 in balancing the growth of sprouts and stems,of which the disharmony may result in disease symptoms.9.Hypothesis of metabolic pathway of witches' broom disease.There may be several interrelated feedback branches in metabolic pathway of witches' broom induced by CK,disorder of any branch would cause different disease symptoms.More branch disorders there are,more serious symptoms would emerge.Among them,3 genes involved in production of folic acid precursor,c72344_g1_i2|m.135613,c62908_g4_i1|m.229979 and c60692_g1_i1|m.185242,may participate in the whole process of the disease metabolism. |
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