Transcriptional Regulation And Dissection Of Genetic Variation Of Secondary Growth In Populus Tomentosa

Wood derived from secondary growth is an important source of plant biomass and accounts for the bulk of renewable biomass worldwide.With the rapid development of social economy,the demand for wood is increasing.Thus,it has been one of the main objectives of forest breeding to select the elite germplasms of trees and improve the yield and quality of wood in order to alleviate the shortage of timber resources.It has been shown that secondary growth and wood formation are complex biological processes,and that tree growth and wood traits are jointly regulated by multiple genes at multiple levels.Therefore,researches on transcriptional regulation of secondary growth ot trees to identify the key regulatory factors atfecting wood formation,and dissection of the genetic basis of grouth and wood traits in trees population are the keys to carrying out marker-assisted breeding of forest trees.This study used the most important commercial tree species(Populus tomentosa Carr.)as the material to systematically investigate the transcriptional regulation of secondary vascular tissues(i.e.vascular cambium,developing xylem and mature xylem)by using RNA-seq.Based on the genome resequencing of 435 individuals in a natural population of P.tomentosa,this study further deciphered the genetic effects(additive,dominant,and epistatic effects)of SNPs within transcriptional regulators underlying lignocellulosic traits.The main results and conclusions in this study are as follows:1.This study identified a total of 4.761 differentially expressed genes in the three comparisons(high vs low biomass)of secondary vascular tissues using RNA-seq.KEGG enrichment analysis revealed 246 secondary cell wall biosynthetic genes(SBGs)enriched in cellulose and xylan and lignin biosynthetic pathways,which might be regulated by transcription factors.such as NAC,MYB and classⅢ HD-ZIP.SNP-based association analysis indicated that 533 SNPs within the SBGs were significantly associated with lignocellulosic traits(P<1.0E-03),each SNP explaining 4.53%-29.82%of the phenotypic variance.Epistasis analysis uncovered a total of 3,576 epistatic SNP-SNP pairs,indicating abundant genetic interactions among the pathway genes.2.By using sRNA-seq.a total of 402 known miRNAs and 24 novel miRNAs were identified in the secondary vascular tissues in P.tomentosa.Among these,147 were differentially expressed in the three comparisons,which might interact with 833 potential targets that were related to vascular development and wood formation.Pearson’s correlation analysis found not only negative but also positive correlations between miRNAs and their cognate target genes,indicating the complex miRNA-mediated regulation.SNP identification showed that the mature regions of miRNAs and target sites were more conservative than their flanking regions(±1kb).Moreover,this study found that SNPs in the precursor regions might affect the stability of secondary structure and miRNAs production;while SNPs in the binding sites of miRNAs and target genes might affect their interactions.SNP-based association analysis identified 498 SNPs within 86 miRNA genes and 655 SNPs in 274 target genes were significantly associated with lignocellulosic traits(P<1.0E-03),explaining 7.9%-27.6%of the phenotypic variance.Furthermore,eQTN mapping revealed miRNA-mediated multi-layered regulatory networks,in which miRNAs can regulate the expression of their downstream SBGs with three regulation models,thus affecting lignocellulosic traits at transcriptional levels.This study provides new insights into the secondary cell wall biosynthetic pathways responsible for wood formation.3.This study has systematically identified a total of 15.691 IncRNAs from the secondary vascular tissues in P.tomentosa.Comparing with Populus mRNAs,these IncRNAs have shorter length and lower sequence conservation and expression levels but higher tissue-specificity.In the three comparisons of secondary vascular tissues,a total of 1,083 differentially expressed IncRNAs were identified,which have 2,143 potential cis-regulated target genes and 2,002 potential trans-regulated targets.Functional analysis and pathway enrichment analysis revealed that these target genes may be involved in secondary cell wall formation.SNP-based association analysis showed that 1.163 SNPs from IncRNAs genes and their potential target genes were significantly associated with lignocellulosic traits(P<1.0E-03),each SNP explaining 1.23%-29.25%of the phenotypic variance,suggesting that IncRNAs may be involved in genetic regulation of growth and wood traits.Epistasis analysis detected a total of 17,656 epistatic SNP-SNP pairs,representing genetic interactions between 11 lncRNAs and 448 target genes,providing supporting evidence for interactions between IncRNAs and their potential targets.4.Analysis of the interactions between miRNAs and lncRNAs revealed that Populus lncRNAs may be involved in niRNA-medited regulation as miRNA prectirsors,potential targets and target mimics.Among these,11 lncRNAs were predicted as the precursors of 15 miRNAs.476 lncRNAs as potential target genes of 213 miRNAs and 28 IncRNAs as endogenous target mimics(eTMs)of 14 miRNAs.Based on this,the proposed miRNA-lncRNAs-mRNA interaction networks were reconstructed,including 223 miRNAs,504 IncRNAs and 89 differentially expressed mRNAs(including 23 SBGs).The results may suggest the complex interactions among these regulators related to secondary cell wall biosynthesis.Combined with the results of the association analysis,this dissertation systematically analyzed the genetic effect of the interaction networks on wood fiber traits,which may enrich the secondary cell wall biosynthetic pathways.