Genetic And Epigenetic Architectures Of Flavonoid Metabolites In Leaves Of Populus Tomentosa

Flavonoids are widely distributed secondary metabolites in plant kingdom with important metabolic functions in different biological processes,such as plant growth and development,reproduction,and stress defense.Therefore,dissecting the genetic regulatory networks underlying flavonoids can facilitate the further exploration of its biological function.Previous studies have shown that flavonoids are subjected to the regulation of a large number of genes with minor genetic effect.Therefore,providing insights into the genetic basis of the formation of flavonoids from the genetic and epigenetic aspects and excavating major genes will greatly promote the understanding of flavonoids biosynthesis.Forest trees are perennial land plants and subjected to the living pressure caused by the constant changes in the external environment,of which flavonoids play an important role in their adaptation.Therefore,forest trees are excellent models for the study of flavonoids biosynthesis.Herein,this study used 300 genotypes of Populus tomentosa collected from the natural distribution regions of China as experimental materials to analyze the flavonoids variation pattern through widely targeted metabolome and further systematically investigate the genetic and epigenetic regulatory networks underlying flavonoids employing multi-omics strategy.The main research contents and results in this study as follows:(1)This study determined 172 flavonoids metabolites and their derivatives through performing widely targeted metabolome using natural population of P.tomentosa.More than 68%of flavonoids are with the coefficient of variation(CV)greater than 0.5,indicating that the high variation level of different metabolites.Pearson correlation revealed that a total of 14,724 pairs of metabolites were with|r|>0.3.Among them,8,250 and 6,474 pairs were positive and negative correlation,respectively.It was found that the flavonoids in the same category tended to have obvious positive correlation,implying the high-level genetic correlation of them.Estimates of heritability showed 69flavonoid metabolites were with the broad-sense heritability(H~2)more than 0.6.Notably,clustering analysis of high-heritability flavonoids clearly separated these natural ecotypes into three different subpopulation which was consistent with the structure and principal complement results calculated by the genome common single nucleotide polymorphism(SNP)loci,implying the high-heritability metabolites can reflecte the adaptation to a certain environment,by which it is feasible to infer the natural population structure.To identify the flavonoids that were targeted by selection in three geographical regions of Populus,this study further calculated Qst of flavonoids traits and estimated inter-population mean Fst among three geographical regions,and found that 83 flavonoids exhibited selection signal among three geographical regions including 19 directional selection and 64 stable selection(P<0.05),suggesting that these metabolites play an important role in the adaptation to different geographical environments of poplar.(2)The mixed linear model(MLM)in EMMAX software was used to perform the metabolome-based genome-wide association analysis(m GWAS)of 172 flavonoid metabolites.As a result,a total of 1,549 significant SNPs for 103 flavonoid metabolites were identified(P<1.01E-07).Among them,282 and 48 significant SNPs showed additive and dominant effect(P<0.01),respectively,and 41 significant loci showed joint additive and dominant effects for flavonoids traits,illustrating that the associated SNPs exhibited various genetic effects.Furthermore,a total of 4,607e QTLs representing 5,295 significant associations were identified for 410 causal genes(P<1.01E-07),including 4,305 trans-e QTLs and 302 cis-e QTLs,which illustrated the distant e QTLs explain a higher proportion of the genetic variance for gene expression.The annotated genes in m GWAS and e QTL mapping were further used to conduct the weighted gene co-expression network analysis(WGCNA),and four co-expression modules and 54 core genes(Connectivity degree?10,weight value>0.1)were identified.A flavonoid biosynthetic network consisting of 30 flavonoid metabolites,48 m GWAS and 14 e QTL annotated genes was constructed in poplar for the first time,which laid a foundation for further analysis of regulatory network underlying flavonoid metabolites in Populus.(3)Epigenome-wide association analysis(EWAS)was performed using the general linear model(GLM)in ewas2.0 software to identify 679 significant associations between 465 SMPs and35 flavonoid metabolites(P<1.26E-08).When compared with m GWAS results,82 and 14metabolites were only identified in the m GWAS and EWAS results,respectively,and 21 flavonoids were detected in both association results,indicating that the formation of flavonoids was controlled differently by genetic and/or epigenetic variation.Expression quantitative trait methylation(eQTM)mapping identified 1,257 eQTMs for 51canditate gene expression,including 299 cis and 1,113 trans associations,suggesting that trans-QTM is more common in regulating gene expression.(4)The causal relation between significant SNPs and SMPs and the expression of candidate genes was examined by the causal inference test(CIT),and thus identified 162 triplets(P<0.001),indluding 84 cis-and 78 trans-effect triplets.These results indicate that both cis-and trans-effect of SNP and SMP play an important role in regulating gene expression.In addition to the interaction effect of SNP-SMP,most SNPs and SMPs have independent effects,which reflects their independent influence on gene expression and phenotypic traits.(5)Finally,the effects of SNP-SMP on anthocyanin metabolite and then the adaptation of the geographical distribution of poplar were thoroughly analyzed in this paper.A total of 123 potential selected genes were firstly identified according to m GWAS and selection sweep results.Therein,the candidate LDOX gene played an important role in the biosynthesis of delphinidin and pelargonidin which showed directional selection in different geographic population of P.tomentosa.Coexpression and e QTL mapping analysis revealed that b HLH121 was a core gene in regulating the expression of LDOX.Meanwhile,SMP in the promoter region and SNP in the downstream region of LDOX exhibited interactions in affecting its own expression,implying that the b HLH121 and LDOX gene play an important role in environmental adaption mediated by genetic and epigenetic variation.This study provided an in-depth analysis of the genetic and epigenetic regulatory networks of flavonoids,which filled the research blank of flavonoids biosynthesis at the population level of perennial trees and provided sufficient genetic resources for forest molecular assisted breeding.