| Photosynthesis is one of the most important chemical reactions of the world,which can use solar energy to convert CO2 into organics,and provide basic energy for plants,microorganisms,animals and human.It is the material bases for almost all lives.Therefore,it could say that without photosynthesis,there would be no lives.However,photosynthesis is restricted by many biological and abiotic factors,resulting in photosynthesis rate far less than the maximum photosynthesis potential.Moreover,the stresses such as drought,heat,salt and alkali occur frequently,which severely limit photosynthesis and thus reduce crop yield and wood product.Therefore,improving photosynthesis and the ability of plants respond to climate change and stresses may be helpful for improving crop yield,wood economic effect.Previous studies showed that transcription factor(TF)could initiate gene expression via binding to the promoter cis-acting elements,and play important roles in photosynthesis regulation.However,the entire regulation of TFs on photosynthesis pathway,the effect of genetic variations within TFs and photosynthesis genes on photosynthesis traits remain to be studied.Therefore,we identified photosynthesis pathway genes of Populus tomentosa Carr.based on the public database,and constructed multi-layer gene regulatory networks(GRNs)of TFs upon photosynthesis genes.And then,we combined DNA resequencing data,metabolome and transcriptome,comprehensively utilizing association genetics to thoroughly elucidate the effect of genetic variations within TFs and photosynthesis genes on photosynthesis traits and systematically exploring genetic regulation of TFs on photosynthesis.The main results and conclusions are as follows:1.This study detected the variations of 23 photosynthesis related traits of 435 individuals of Populus tomentosa,including five photosynthesis physiology traits,eight leaf morphology traits and ten photosynthesis enzymatic traits.Results showed that the entire variations of photosynthesis enzymatic traits are larger than photosynthesis physiology traits,and variations of leaf morphology traits are the smallest.Detection of phenotypic distribution patterns revealed that 8 traits distributed normally,9 could convert to normal distribution via log transformation and the rest 6 could transform to normal distribution via Jonson transform or normalized by SPSS,indicating that these traits may be used for association analysis directly or after transformed.Further examination of the phenotypic and genotypic correlations between these traits identified 69 phenotypic correlations and 66 genetic correlations,and a relative strong correlation between phenotypic and genetic correlation(r=0.87,P?0.002),indicating that photosynthesis traits may be controlled by genetic factors.Moreover,traits within the same categories tended to be more closely related(P=8.07 × 10-7,Kolmogorov-Smimov test),and for the positive correlations,there also exhibited larger correlations within the same categories than between categories,but no obvious differences for negative correlations were found2.This dissertation thesis identified 146 photosynthesis genes in Populus tomentosa,including 96 photosynthesis light reaction genes,52 photosynthesis carbon assimilation genes based on public databases.Examination of gene expression patterns within 11 various tissues and organs of poplar(vertex,stipe,expanding leaves,immature leaves,mature leaves,bark,cambium,phloem,immature xylem,mature xylem and root),showed that the vast majority of genes mainly highly expressed in mature and expanding leaves,indicating these genes mainly functioned in leaves.17,051 SNPs were identified within these 146 genes among the association population,of which 12,426 were common(MAF? 0.05,missing<20%).The nucleotide diversity and linkage disequilibrium(LD)were relatively low,with LD decayed to 0.2 within 4,174 bp,indicating gene-based association mapping is feasible3.Exploration of cis-acting elements within 96 photosynthesis light reaction genes' promoters identified 118 motifs coexisted within 80%promoters,mainly involved in light response,stress response and phytochrome response.Moreover,346 TFs from 27 TF families were identified interacting with these motifs.This study constructed a three-layer GRN involving 64 photosynthesis genes and 54 TFs via BWERF,consisting 404 gene pairs.Genotype-phenotype association identified 304 SNPs from 88 genes associated with 12 photosynthesis traits(P ? 1 ×10-4),explaining a mean phenotypic variation of 18.82%.The majority of significant SNPs were located within non-coding regions,with intron region harboring the most SNPs(32.57%),followed by promoter region(29.93%),indicating non-coding regions may play a regulatory role.Moreover,119 significant SNPs were from 34 TFs,indicating variants within TFs may have a general role on photosynthesis traits.And 26 TFs were associated with the same traits with their potential targets of 35 genes,indicating these TFs may be involved in the same biological pathway with their targets,thus affecting the same traits.Epistasis analysis identified 440 SNPs within 95 genes composing 770 SNP-SNP epistatic pairs upon 13 photosynthesis traits,explaining a mean variation of 0.31%.This study identified that epistasis mainly occurred between non-significant SNPs,indicating that epistasis may be more effective in identifying minor effect loci.Moreover,the epistasis mainly identified within different families(95.59%),indicating that epistasis analysis could be used to identify interactions within a pathway,and may be helpful in understanding biological pathway and genetic homeostasis.Of the significant genes being epistasis with other genes,38 were TFs,of which 2 TFs simultaneously be the key genes of Ci,Pn,Trmmol and LWC,indicating TF may impact photosynthesis traits via being epistatic to other genes.eQTL analysis identified 175 SNPs within 138 genes associated with 104 eGenes,consisting 2830 associations,of which 942 pairs were from TF-target pairs,further indicating that TF may be important in regulation of photosynthesis genes.Results provides important basis for TF regulating photosynthesis light reaction genes in Populus.4.Promoter analysis of 52 photosynthesis carbon assimilation genes identified 706 motifs and 326 TFs.A three-layer GRN composed of 40 TFs and 46 photosynthesis carbon fixation genes was constructed using BWERF method,each layer contained 46 photosynthesis genes(the first layer),25 TFs(the second layer)and 15 TFs(the third layer).Genotype-phenotype association identified 248 SNPs within 72 genes associated with 11 photosynthesis traits.Of the 201 gene regulatory pairs identified by BWERF,77 TF-target pairs were associated with the same traits,indicating these TFs and target genes may affect phenotypes via the same mechanisms.eQTL analysis identified 1,851 association signals about 50 eGenes,and 14 eQTLs within 7 TFs associated with their target gene expression traits,of which variations within genes from 7 TF-target pairs associated with the same traits.This study constructed a gene regulatory network of poplar and provided new insights for elucidating the mechanisms of photosynthesis in Populus.5.This thesis selected genes within the GRN identified by BWERF,that were also identified by association,epistasis and eQTL analysis as key genes,identifying 63 TFs and 81 photosynthesis genes.Gene correlation analysis of various tissues identified 41 TFs and 60 photosynthesis genes coexpressed.Metabolic analysis identified 1705 SNPs within 82 genes associated with three photosynthesis metabolic traits,explaining a mean variation of 11.58%.Of the significant genes,33 were TFs,indicating most of the key TFs identified in this study may generate impact on photosynthesis metabolic traits,further validated these TFs involved in photosynthesis regulation.The results provided new insights in identifying regulatory relationships of TFs upon photosynthesis genes and genetic regulatory mechanisms of TFs upon tree photosynthesis.This thesis may provide important theoretical values for TFs involved marker-assisted breeding for photosynthesis improvement of forest tree. |
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