| Mahaleb ‘CDR-1' is suitable for cultivation in north area of China because of strong resistance ability to crown gall disease,cold,and salt and alkaline.Dwarf rootstocks is conducive to density cultivation and the early fruit,but the dwarf characteristics of p.mahaleb has not been solved.Organisms involves in regulating gene expression by regulating the methylation status of genes,especially the promoter region and coding regions.Generally speaking,demethylation can activate the gene expression,and methylation can suppress gene activity.It has been proved that the methylation status of dwarf plants and normal plants were different.MSAP is one of the most widely used tools for genome-wide methylation detection,and the MSAP technique can identify difference of DNA methylation status from individual plant.In this study,we choosed 25 dwarf plants from self-crossed progeny of p.mahaleb 'CDR-1'(denoted as dwarf group),and 25 normal plants(denoted as semi-dwarf group).MSAP fingerprints were obtained to analyze the methylation level and pattern,to investigate the relationship between dwarf traits and genomic methylation.The main results of this dissertation are as follows:1.The MSAP technique were optimized,including DNA digestion,pre-selective amplification and selective amplification.A total of 15 pairs of primers were selected from the combination of the 64 pairs of primers,and the MSAP fingerprints were obtained about dwarf group(25 individuals)and semi-dwarf group(25 individuals).2.15 pairs of primers detected 9021 loci between the dwarf group and semi dwarf group,there were an average of 180 loci per individual;685 were semi-methylation loci,there were an average of 14 loci per individual,and the rate was 7.59%;2657 were full methylation loci,there were an average of 53 loci per individual,and the rate was 29.45%.Thus,the the main mode of methylation of p.mahaleb on "CCGG" sequence was double-chain methylation.The total loci of semi-dwarf group were 4577,hemi-methylation loci were 336,and full methylation loci were 1274.The total loci of dwarf group were 4444,hemi-methylation loci were 349,and full methylation loci were 1383.T-test and one-way ANOVA showed that the level of full methylation and total methylation in semi-dwarf group were extremely significantly different than that in dwarf group,and the level of hemi-methylation was significantly different.Meanwhile,the level of methylation in dwarf group was higher than that in semi-dwarf group.3.By analysis of the methylation pattern,the results showed that 85.53% of amplified loci were methylation polymorphism loci in semi-dwarf group,and 89.31% of amplified loci were methylation polymorphisms loci in dwarf group.That showed the polymorphism of dwarf group were highter than that of semi-dwarf group.The polymorphic loci of two groups occurred mainly in double chain methylation loci and hypermethylation loci.Further analysis of the types of polymorphism showed that the frequency of A4 type was higher in dwarf group than that in semi-dwarf group,and the frequency of A2 type was lower in dwarf group than that in semi-dwarf group,indicating that the locus of hypermethylation in dwarf group was higher than that in semi-dwarf group.The research indicated the correlation between dwarf characteristic of p.mahaleb and genome methylation modification from the differences in DNA methylation levels and patterns between dwarf group and semi-dwarf group.The results might provide theoretical support for further breeding of cherry dwarf rootstock. |
PDF Full Text Request