Analyses Of Transcriptional Regulation And Allelic Variations Involved In Reaction Wood Formation In Populus Tomentosa

Biological transcription regulation is a complex process which is regulated under multi dimensions,genes,and factors.It is precisely and accurately regulated in time and space from DNA level,transcriptional level and post transcriptional regulation level,and so on.Wood property is a quantitative trait.The biological regulation of wood formation is a complex process.Understanding of regulation mechanism and genetic variation of wood formation,will not only help us to understand the molecular mechanism of wood formation,and to improve quality of wood traits.Therefore,this study took the poplar(Populus tomentosa Carr.)which is the important tree species in China as the research object,tried to reveal the regulation and genetic variation of wood formation using the tension wood model combined with a natural populations of P.tomentosa by RNA-seq,BS-seq,Degradation-seq and Resequencing.The main results and conclusions in this study as follows1.The results of sequencing have found 361 genes were differentially transcribed(log2FC≥1 or≤-1,P≤0.05 and FDR≤0.05)between tension wood(TW)and opposite wood(OW),2,658 differed between OW and normal wood(NW).and 2,417 differed between TW and NW,indicating that NW differs significantly from the wood in branches.The differentially expressed genes included 97 encoding transcription lactors(TFs),40 involved in hormone signal transduction,33 in lignin biosynthesis,21 in flavonoid biosynthesis,and 43 in cell wall metabolism,including cellulose synthase,sucrose synthase,and COBRA.More 39 TF genes in TW and 50 in OW showed more than 4-fold lower transcript levels in NW compared with TW or OW.indicating that TF abundances differed dramatically in different wood types and may have important roles in the formation of reaction wood.In addition.28 transcripts of 33 differentially expressed genes involved in lignin biosynthesis were more abundant in OW compared with TW.consistent with the higher lignin content of OW.Genes involved in cellulose biosynnthesis have different patterns of expression in NW,TW and OW,showing that the genes involved in reaction wood formation process and there are divergences in their expressions and functions.After detecting the relationship between the significantly expressed genes by co-expression analysis,218 pairs of genes that were significantly compressed were found.We constructed two transcriptomic networks for the regulation of lignin and cellulose biosynthesis,including TFs,based on the co-expression patterns of different genes(48 genes).2.A total of 3 17 known and 35 novel miRNAs were identified using the small RNA-seq in a tension wood system.Among these,21 7 miRNAs differentially expressed,and identified 1,196 candidate target genes which involved in many processes,such as macromolecule biosynthetic process and regulation of gene expression.Degradome sequencing confirmed 60 of the target sites.Also,72 miRNA-target pairs showed significant co-expression.GO term analysis showed that most of the genes in the co-regulated pairs involved biological regulation.Genome re-sequencing found 5,383 common single nucleotide polymorphisms(SNPs)(frequency≥0.05)in 139 miRNAs and 3 1,037 SNPs in 819 target genes,which could contribute as much as 39.74%phenotypic variation.Single-SNP association analyses identified 1 89 miRNA-target pairs associated with the same traits.Multi-SNP associations found 102 epistatic pairs associated with phenotypic traits.Furthermore,a reconstructed regulatory network contained twelve significantly co-expressed pairs,including eight miRNAs and nine targets associated with traits.Lastly,both expression and genetic association showed that miR156i,miR156j,miR396a and miR6445b involved in the formation of tension wood.3.Whole-genome bisulfite sequencing showed that OW had more methylated cytosine(8.1%)than TW(6.1%).Combing with RNA-sequencing,DNA methylation affected gene expression varied transcribed regions in different ways.We identified 60,654 differently methylated sites(DMSs)(P≤0.05)between TW and OW.Analysis of 15,220 DNA sequences containing 100 bp flanking the DMSs identified 5,566 common SNPs(frequency≥0.05).Single-SNP(P≤0.05 and Q≤0.05)which could contribute 19.11%phenotypic variation in average and multi-SNP(P≤1×10-7)association studies detected 141 and 290 significant associations,respectively,between these SNPs and wood properties and tree growth.Thus.DNA methylation significantly differed between TW and OW,and SNPs in DMSs and their flanking sites associate with wood formation in P.tomentosa.This thesis aims to improve the breeding of tree with high quality wood properties and reveal the transcriptional regulation and genetic variation in wood formation.Thus,the genomics,molecular heredity and population genetics theory and technology were used to identified mechanism of transcriptional regulation involved in wood formation in P.tomentosa by reaction wood model.The networks between TFs and key genes involved cellulose and lignin biosynthesis were identified including Pt-MYB156.And the differentially expressed miRNAs were found and revealed miRNAs such as miRl 56i involved in the regulation in reaction wood.Association studies found 1614 SNPs in mRNAs and their target genes associated with traits.The results of sequencing found the DNA methylation levels in TW was lower than OW and association studies detected 134 SNPs significantly associated with wood properties traits in the DMSs and their flanking sites.This study provided the basis for quality improvement of tree breeding and cultivation of new varieties.