Analysis Of DNA Methylation Of Multicolor Petal Of Paeonia Suffruticosa Based On MSAP

Tree peony(Paeonia suffruticosa Andr.)is native to China and and its flower color is usually charming and changeable.Among the numerous flower color phenotypes of tree peony,multicolor is relatively rare and has great ornamental value.However,the real mechanism of the formation of multicolor flowers in tree peony has not yet been revealed.It is of great significance to explore the mechanism of multicolor petals for guiding regulation and cultivating new multicolor flowers cultivars in P.suffruticosa in the future.In this study,the multicolor cultivar ‘Shima Nishiki' and red petals cultivar ‘Taiyoh'(‘Shima Nishiki' is a budded cultivar of ‘Taiyoh')with flower color phenotype stably inherited was selected as study object.The methylation sensitive amplification polymorphism(MSAP)reaction system was used to analyze DNA methylation level and pattern of ‘Shima Nishiki'and ‘Taiyoh' two cultivars,red and pink petals in‘Shima Nishiki' respectively.The DNA methylation differential bands were recovered,cloned and sequence aligned to screen for methylated genes.It is expected to found out the formation of multicolor flowers from the perspective of DNA methylation.The main findings are as follows:1.To determine a appropriate method for genomic DNA extraction.Genomic DNA was extracted from petals of tree peony by modified CTAB method and kit method,and compared the results.The results show that although the steps in modified CTAB method are tedious and time-consuming,the extraction result is better than the kit,the DNA quality is high,and there is no dragging phenomenon,which is more suitable for subsequent experimental analysis.2.Optimize the MSAP reaction system of P.suffruticosa.Explored and established a set of MSAP technology system of P.suffruticosa.The optimal digestion time was determined for 1 hours,the enzyme-linked product was diluted20-multiple,and 31 pairs of primer sets with clear,stable and great polymorphic amplified bands were screened from 100 primer sets.3.Preliminarily proved that the lower level of DNA methylation were related to theformation of multicolor in P.suffruticosa.The genomic DNA of the red and pink petals of ‘Shima Nishiki' were analyzed for methylation levels during the soft bud stage,initial bloom stage,and full bloom stage.A total of 4035 clear and identifiable bands were amplified from 31 pairs of primers,of which 1983 methylation sites,accounted for 49.14%.Among all the amplified sites,the methylation level of red petals was detected between 47.55% and 58.45%,and that of pink petals was44.42%-47.54%.The methylation level of pink petals were always lower than that of red petals,especially in the initial bloom stage,the difference was significant.The results indicate that lower levels of methylation may be the cause of the formation of multicolor flower in‘Shima Nishiki'.4.Initially clarified the higher proportion of demethylation patterns were related to the formation of multicolor in P.suffruticosa.Analysis of genomic DNA methylation patterns of red and pink petals revealed that four methylation patterns of non-methylated changes(type A),remethylated(type B),demethylated(type C),and undefined patterns(type D,the proportion were small and weren't included in the calculation)in ‘Shima Nishiki'during soft bud stage,initial bloom stage,and full bloom stage.Type A occurred in three different flowering periods with a higher proportion of 76.80%,57.11%,and 83.14%,respectively.It can be seen that the pattern of non-methylated changes were dominant.Types B and C are polymorphic patterns,among of them,type B in the three stages were 11.27%,11.49% and 8.31%,respectively,type C were11.93%,31.40% and 8.55% in the three stages.It was found that the proportion of type C was always higher than type B in the same stage,especially in initial bloom stage,the difference was significant.This result indicates that a higher proportion of the demethylation pattern were related to the formation of multicolor in P.suffruticosa.It were speculated that some key genes were demethylated in the petals,which promoted the gene expression,which eventually led to differences in flower color.5.To identified one gene,PsbHLH1,which negatively regulates the synthesis of anthocyanins of ‘Shima Nishiki'.A total of 85 methylation-specific bands were recovered,ligated,transformed,and sequenced.One gene related to anthocyanin synthesis was obtained by BLAST comparison,which was PsbHLH1.Through its expression analysis at different stages of ‘Shima Nishiki',it was found that the relative expression in pink petals was always higher than red petals,indicating that PsbHLH1 has a negative regulatory effect on the synthesis of anthocyanins of‘Shima Nishiki'.