Identification And Genetic Effect Analysis Of Cytokinin Transcriptional Regulatory Pathway Genes In Populus Tomentosa

Forest is the largest terrestrial ecosystem on earth,with important ecological value and productive value as a part of the global biosphere.With the rapid development of human society and economy,the demand for yield and quelity of timber is increasing.Therefore,the main goals of forest breeding at present stage are to improve the propagation efficiency,shorten the breeding cycle and improve the quality of wood by means of modern biotechnology.Cytokinins,one of the six recognized plant hormones,play important and broad-spectrum regulatory roles in plant growth and development.In trees,cytokinins can affect the growth of roots and stems,the development of vascular cambium and leaf senescence.However,the specific mechanism of these effects still needs to be further explored and revealed.Therefore,it is of great signif icance to systematically study the regulation mechanism of cytokinin metabolism and the effects on growth and wood quality of forest trees.Thus,this study used Populus tomentosa population as the material to study the natural variation of plant hormone metabolites systematically by using the LS/MS-MS technique.High throughput sequencing and bioinformatics methods were used to systematically study the physiological,photosynthesis and molecular level variation patterns of poplar in response to cytokinin.Further,cytokinin pathway genes of Populus tomentosa were identified based on the public database,the related transcription factors were predicted using bioinformatics methods.Cytokinin related candidate genes databset was constructed,including of cytokinin-responsive genes,cytokinin pathway genes and transcription factor genes.The regulation network of cytokinin metabolism was constructed by means of multi-omics to analyze the genetic regulation effect of candidate genes on plant hormone metabolism.Finally,association mapping was conducted to analyze the effects of candidate genes on growth and wood quality of poplar.AS E analysis was used to uncover the intrinsic mechanism of dominant effect.This study provides a theoretical basis and technical support for the research on the genetic regulation of cytokinins and molecular marker-assisted breeding in forest trees.The main results and conclusions are as follows:1. Population determination of plant hormone-related metabolites in 435 plants was carried out by means of a broad targeting metabolome.15 plant hormones metabolites were detected,including indole carboxylic acid,kinetin 9-riboside,trans-zeatin N-glucoside,trans-zeatin 9-O-glucoside,indole 3-acetic acid,N⁶-isopentenyladenine,trans-zeatin,cis-zeatin,?+?-dihydrojasmonic acid,1-naphthylacetic acid,salicylic acid O-glucoside,?+?-jasmonic acid,salicylic acid,gibberellin A3,N-[?-?-jasmonoyl]-?L?-isoleucine.The detection of coefficient of variation and the estimation of heritability showed that these metabolites were greatly influenced by genetic factors,especially cytokinins.Furthermore,Pearson's correlation test of different metabolite traits was carried o ut by statistical method,revealing the potential interaction between cytokinins and various metabolites.2. The experiment of exogenous cytokinin?6-BA?treatment was carried out on the clones of annual Populus.The physiological and photosynthetic indexes of Populus tomentosa were determined after 6-BA treatment.The results showed that the total protein content,sucrose phosphate synthase?SPS? activity,peroxidase?POD?activity and malondialdehyde?MDA?content were significantly affected within 24 h after 6-BA treatment.The physiological changes were the most obvious at 6 h after 6-BA treatment.Four weeks after treatment,the Pn,Gs,Tr were significantly improved in P.tomentosa.These results showed that 6-BA could signif icantly affect the physiology and photosynthesis traits of P.tomentosa.3. By using RNA-seq,this study identified 501 6-BA-responsive genes.Pathway enrichment analysis revealed that these 6-BA-responsive genes were related to catalytic and metabolic processes.In addition,6,283 P.tomentosa lnc RNAs were identified at genome-wide,which exhibited shorter length and lower expression than the protein-coding m RNA.A total of 262 lncrnas were found in response to 6-BA,and 210 potential cis-target genes and 214 potential trans-target genes were predicted.Functional annotation and pathway enrichment analysis revealed that the potential target genes of these lnc RNAs may participate in a variety of metabolic and redox processes.Small-RNA sequencing technology was used to detect 24nt-siRNA.The results showed that the diversity and abundance of 24-nt siRNAs decreased sharply under 6-BA treatment.A total of 15,793 24nt siRNA clusters were found in response to 6-BA,most of which existed in the intergenic regions.Allelic specific loci were identif ied by RNA-seq.A total of 102,819 loci were found to have allelic imbalance expression variation in response to 6-BA,most of which were located in the exon regions.The results showed that allele-specific expression was widespread.The high-throughput methylation sequencing technique was used to detect 566 differentially methylated regions in response to 6-BA.The transcriptional regulation of DNA methylation on genomic elements was analyzed by expression level analys is and ASE level analysis of genomic elements in DMRs.The results showed that the difference of ASE level of protein-coding gene decreased,while the difference of allele of lnc RNAs increased under 6-BA treatment.4. Based on the known information in a public database,69 cytokinin pathway genes and 121transcription factors were identif ied in the poplar genome.A candidate gene set composed of cytokinin response gene,cytokinin pathway gene and its related transcription factors was constructed.The expression patterns of candidate genes under 6-BA treatment was detected.The results showed that 86.78%of the candidate genes responded to the external 6-BA treatment,indicating its important role in cytokinin synthesis,metabolism and signal transduction.Using the resequencing data,a total of 37,916 common SNPs?MAF?0.05,missing?20%?were detected.The level of nucleotide polymorphism and linkage imbalance was relatively low,and r²decreased to below 0.2 at a distance of 735bp,indicating that the association mapping based on candidate genes was feasible.5. A total of 37,916 genome mutation sites,271 gene expression levels,and 15 phytohormone metabolite contents were obtained.Using association mapping,eQTL and co-expression analysis methods,such as found 954 signal s ites that regulate and control four metabolites,2,634,581 eQTL loci,and 122 groups of metabolites content was signif icantly associated with gene expression;In addition,SNP shared by association mapping and eQT L was used to construct the networks of metabolite-SNP-gene,including two metabolites,146 SNPs and ten genes.To further explore the leader SNP associated with the two metabolites,the results showed that leader SNP G547₁15 of Pto WRKY42 gene regulated the expression of Pto UGT76C1 in poplar as a trans-eQTL and affected the content of trans-zeatin N-glucoside?ZNG?.Pto UGT76C1 coding protein could catalyze the degradation of trans-zeatin to trans-zeaxanidin,which affected the metabolism of endogenous cytokinin.In addition,Pto WRKY49 gene has a leader SNP G501₅,which regulates the expression of Pto SYP121 gene,which encoding synaptic fusion protein as trans-eQTL and affects the content of indole-3-carboxylic acid?ICA?.Under the action of ICA,this gene can promote the hydrolysis of starch into corpus callosum to resist the infection of humic bacteria and play an important role in plant resistance to biological stress.6. Association analys is was used to analyze the regulatory effect of genetic variation of candidate genes on growth and wood quality of poplar.152 SNPs were identified and 182 pairs of associations were formed with seven growth and wood quality traits?DBH,V,HEC,HC,AC,LC,FW??P<1.32E-6?.24 SNPs showed prominent dominant effect?d/a>2?.ASE analysis was used to analyze the dominant effect mechanism,it was found that the DMR_Chr02₂4663250 regulated by siRNA_cluster₃2657 was semi-methylated,mediating the allele-specific expression of TCONS₀0053467-MIK2 homologous gene,which played an important role in the dominant effect of TCONS₀0053467 on DBH and volume.