| The analysis of the growth advantage mechanism of polyploid plants has become a research focus in the field of forest breeding.At present,the analysis of the molecular mechanism of the advantage of poplar triploid growth is focused on the transcription level.In this study,the genome-wide bisulfite methylation sequencing(WGBS)technology was used to analyze the two populations that were generated by the same female(P.pseudo simonii × P.nigra var.italica)and male(P.× beijingensis)parent.Analyze the apical bud and leaves of the triploid and diploid progeny to explore the changes in DNA methylation patterns after chromosome doubling;combine transcriptome and small RNA sequencing to explored the influence of triploid DNA methylation mutations on gene expression regulation network,and combined with the verification results of transcription factor overexpression,preliminary revealed the epigenetic regulation mechanism of poplar allogeneic triploid vegetative growth dominance.The main results were as follows:(1)There are significant differences in the gene expression patterns of the apical buds and leaves between the triploid and diploid poplars.Transcriptome sequencing analysis was performed on triploid and diploid apical buds and leaves with different leaf positions.The Impulse DE2 method was used to identify 9875 differentially expressed genes(DEGs)in apical buds and different leaf positions.According to the trend of differentially expressed genes with leaf development,these can be divided into four types.Among them,there are 83 genes with high apical bud expression and low expression with leaf development with no significant changes,which are mainly concentrated in the promotion of cell growth and division,amino acid synthesis and other related pathways.Including genes such as ARF,EXPA,HB12;89 genes with low apical bud expression and high expression with leaf development without significant changes,mainly concentrated in photosynthetic carbon fixation,starch-sugar metabolism and other related pathways,including HEMA1,LHCB,SUS and other genes;2336 differential genes that are highly expressed in apical buds and gradually down-regulated with leaf development,and 2443 differential genes that are lowly expressed in apical buds and gradually up-regulated with leaf development,mainly concentrated in plant hormone signal transduction,carotenoids,porphyrins and chlorophyll Anabolism,leaf senescence and other related pathways,including GRF,SAUR,SAG,WRKY and other genes.(2)There are significant differences expression in lnc RNA at apical buds and leaves between the triploid and diploid poplars.Long non-coding RNA directly regulates genes through cis-or transregulation to promote triploid growth.Through the analysis of transcriptome data of apical buds and different leaf strand-specific paired sequencing,17,601 long non-coding RNAs(lnc RNA)were identified,of which 1692 lnc RNAs were differentially expressed involved in the cis-or trans-regulation of 2103 m RNAs.The trend of DE-lnc RNAs could be divided into four types,among which 98 lnc RNAs were highly expressed in terminal bud and low expressed with leaf development without significant change,mainly for promoting cell growth and division and related lnc RNAs,such as auxin and cytokinin.Including XLOC＿023458 and XLOC＿012748.There were 157 genotypes of lnc RNAs with low expression in terminal bud and high expression with leaf development without significant change,which were mainly related to photosynthesis,carbon fixation and starch-sugar metabolism,including XLOC＿011463,XLOC＿069851,etc.There were 345 differentiated lnc RNAs that were highly expressed in terminal bud and gradually down-regulated with leaf development,mainly to promote carotenoid,anthocyanin,chlorophyll synthesis and other related lnc RNAs,including XLOC＿023645,XLOC＿022041,etc.There were 296 differentially expressed lnc RNAs with low expression in terminal bud and gradually up-regulated with leaf development,which were mainly related to hormone signal transduction,glycogen/glycogen genesis and chlorophyll metabolism,such as XLOC＿009033 and XLOC＿022592.(3)The lnc RNA in the triploid leaves of poplar indirectly regulates gene expression through mi RNA,resulting in no dose effect on the terminal bud and leaf mi RNA.Degradome analysis determined that there are 99 mi RNAs and 452 m RNAs in the leaves of Populus triploid,consisting of 575 pairs of mi RNA and m RNA targeting relationships,of which 70 conserved mi RNAs are differentially expressed.Correlation analysis found that vegetative growth-related mi RNA-m RNA target gene pairs has no significant negative correlation.Sequencing analysis found that there are endogenous competitive binding elements(MREs)of mi RNAs on 120 differentially expressed lnc RNA sequences.Differential lnc RNAs bind to the mature mi RNA sequences through base complementation,weakening or even eliminating the dose effect of mi RNA expression.There are 271 regulatory relationships of DElnc RNA-mi RNA-DEGs were identified,and these differentially expressed lnc RNAs were significantly positively correlated with the expression of DEGs.Lnc RNAs indirectly regulate genes expression related to endogenous hormones,chlorophyll synthesis and senescence,photosynthesis and carbon metabolism through mi RNA,significantly promoting leaf growth,chlorophyll synthesis and photosynthesis and delaying leaf senescence in triploid.(4)There are significant differences in the DNA methylation variation patterns of the apical buds and leaves of poplar triploids.Whole genome methylation sequencing(WGBS)was performed on three representative tissue parts of triploid apical bud,5th leaf position and 25 th leaf position,and 6522,6425 and 6605 differential methylation sites were obtained respectively.The methylation levels of all C sites and three sequence contexts(CG,CHG,CHH)of the triploid poplar are about 11.69%,42.05%,29.81%and 4.71%.In the whole genome,the DNA methylation level in the region where the si RNA is located is significantly higher than that in other regions,and the expression of si RNA is positively correlated with the DNA methylation level at the location,indicating that si RNA mediates de novo DNA methylation through the Rd DM pathway.In the whole genome,the DNA methylation level of the region with more genes is lower,the density of transposon is positively correlated with the methylation of the region where the transposon is located.The methylation level of triploid is higher than that of diploid in the CG and CHG context,and m CHH is lower than diploid.(5)The DNA methylation variation in the promoter region of the triploid of poplar negatively regulates the expression of lnc RNA and m RNA.In the apical buds,genes involved in cell division and affecting endogenous replication of cells(ARFs,HB12)decrease in methylation level and increase in expression;in the fifth leaf position,they participate in anthocyanin,carotenoid synthesis,chlorophyll synthesis and light Capture-related genes(PORA,HEME,HEMB)decrease in methylation level,increase expression,and increase photosynthetic carbon assimilation ability;at the 25 th leaf position,leaves respond to abscisic acid and salicylic acid genes(WRKY,SAGs)methylation level Increase,decrease expression,delay chlorophyll degradation and leaf senescence.In summary,there are wide variations in the DNA methylation of triploids,directly negatively regulate gene expression in the gene promoter region;or negatively regulate the expression of lnc RNA,directly affect target gene expression through cis and trans regulation,and through competition Sexual binding eliminates mi RNA dose effects and indirectly regulates mi RNA target genes.DNA demethylation affects the target genes ARF,HB12 and GRF5 of lnc RNA and mi RNA;hypermethylation mutation affects the target genes TCP4,PUB17,SAGs,etc.of lnc RNA and mi RNA.The auxin and cytokinin that are positively related to vegetative growth accumulate at a high speed,and the content of ethylene,abscisic acid,and salicylic acid that are negatively related to vegetative growth decreases,leading to faster cell division and growth of triploid leaves and the leaves become larger.Increasing chlorophyll content,slowing down the degradation of chlorophyll,enhancing the ability of light contraction,can accumulate more sucrose and starch,etc.,which has obvious growth advantages. |
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