| Potato(Solanum tuberosum L.)is an important food crop and economic crop.The production of virus-free seed potato requires the cultivation of tube tubers.The tuber bearing ability of potato in vitro was mainly affected by environmental factors,endogenous hormone levels and genetic factors.DNA methylation is an epigenetic modification that plays an important role in plant growth and development.DNA methylation has plasticity and heritability.Under the stimulation of environmental factors,the methylation patterns and levels of potato genome will change accordingly.It has been reported that DNA demethylation reagent treatment affects the growth and development of potato tubers.However,whether DNA methylation affects tuber formation in potatoes and how it affects tuber formation have not been reported.In this study,we studied the effects of methylation on tuber formation in potato in vitro,potato genome DNA methylation patterns are analyzed before and after tuber formation under photoperiod condition,genome-wide screening of differential methylation sites under 8 h/d and 16 h/d photoperiod condition,analyze the methylated status of whole genome,screening differentially methylated genes were related to differential methylated sites involved in photoperiod induce tuber formation in potato.The main results are as follows.1.Demethylation reduced the initial tuber formation time of potato tubers and improve the potato formation ability of test-tube plantlets.In vitro seedlings of E20,E26,E108 and E3 genotypes treated with demethylation reagent Zebularine(Zeb),The phenotypes of four genotypes were identified under 8 h/d and 16 h/d photoperiod conditions respectively.We found that Photoperiod-sensitive E26 and E3 genotypes were significantly earlier than those of control group after demethylation treatment under short-day induction.However,the treatment group and the control group did not produce potatoes under the condition of long sunshine without induction.In addition,comparing the ability of tuber-forming in vitro between the treatment group and the control group,it was found that the demethylated genotype materials E26,E108 and E3 all showed higher tuber-setting rate,while the nontuber genotype material E20 remained non-tuber-setting after demethylation treatment.2.DNA methylation modification of potato genome responds to changes in photoperiodic conditions.MSAP(methylated sensitive amplification polymorphism)was used to detect the methylation pattern and degree of the whole genome in the process of potato tuber formation induced by photoperiod.The results showed that the DNA methylation level of potato genome increased first and then decreased in response to photoperiod change from the second week to the third week,and the change was most obvious from the first week to the second week.The overall situation from the first week to the fifth week showed that the DNA methylation level of the three genotypes decreased on average.3.There are distinct differences in global DNA Methylation levels of photoperiod sensitive materials in potato.The genomic DNA methylation sites of E20,E26,E108 hybrids with different photoperiod phenotypes were sequenced by using methylrad technique.The distribution data of the whole genome DNA methylation sites of the three genotypes were obtained.Genomic DNA methylation sites of E20,E26 and E108 genotypes with long and short day photoperiod were compared.The results showed that the differences among the three genotypes were greater than that of the same genotype under long and short day photoperiods;E108,which had both long and short day photoperiod,had potato in long day photoperiod and short day The methylation rates of genome were the lowest under photoperiod,which were 22.06% and 17.96% respectively.4.DNA methylation sites in response to photoperiod vary greatly in potato photoperiod sensitive materials.The methylation sites of the three genotypes before and after the photoperiod treated were analyzed,and the results showed that the number of(3387)decreased-methylation-level sites in E20 was more than the number of increasedmethylation-level sites(1608).The number of(1492)decreased-methylation-level sites in E26 was approximately equal to the number of(1424)increased-methylation-level sites.The number of decreased-methylation-level sites in E108(1896)was less than the number of increased-methylation-level sites(2884).Venn diagram analysis was performed on the different sites before and after the treatment of the three genotypes.The common cg-sites were 14 and the common CHG sites were 10,indicating that the methylation of the three genotypes was significantly different.5.Genes involved in photoperiod regulation of potato tuber formation and regulated by DNA methylation were screened.By comparing the methylation levels in gene regions,the differentially methylated genes of three genotypes were screened out.By excluding the differentially methylated genes common to E26 and E20,472 differentially methylated genes related to photoperiod tuber formation were obtained.Further analysis of GO and KEGG enrichment showed that E26 was enriched into the circadian rhythm pathway which was important for photoperiod tuber formation,and then 7 genes modified by DNA methylation under photoperiod conditions were screened.By detecting the effects of demethylation reagents on the expression of these genes under short-day conditions,three genes related to DNA methylation and photoperiod regulation of potato tuber formation were screened.The candidate genes include: Phytochrome?F?Casein kinase and DNAdirected RNA polymerase III.Based on the analysis of gene methylation Island(CGIS)and the molecular metabolic pathway of photoperiod regulating tuber formation,we proposed the possible mechanism of epigenetic modification in potato tuber formation.In this study,the effect of DNA methylation on potato in vitro was clarified,and the basic characteristic information of potato whole-genome DNA methylation was obtained by sequencing for the first time.Based on the sequencing results,we screened out the DNA methylation modifier associated with cycle-induced tuber.By analyzing the methylation changes of these genes before and after treatment,the general pattern of the phenotypic response to photoperiodic changes of the potato can be given.The results of this study enrich the theory of epigenetic modification of potato tuber formation,reveal the epigenetic modification genes that may exist in the process of potato tuber formation,and provide a new perspective to understand and understand the mechanism of potato tuber formation. |
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