DNA Methylation During Seed Maturation And Germination Of Barley

Cytosine methylation has been proved to be an important factor in the epigenetic regulation of gene expression. In this study, we reported the analysis of cytosine methylation during seed development and germination in barley cultivars Steptoe and Morex (Steptoe, a 6-row spring feed cultivar with high levels of dormancy; Morex is 6-row malting cultivar with little dormancy.) using MSAP. The results were outlined as following.1. Genome-wide changes in cytosine methylation level of various DNA samples representing different stages during maturation to germination in seeds of barley cv Morex and Steptoe were detected. A total of 4654 and 4592 MSAP fragments from Steptoe and Morex were obtained by 95 pairs of selective combination primers, respectively. The number of methylation sites was 2777-2894 in Steptoe and 2784-2882 in Morex. It revealed that methylation levels of total amplified sites in 12 seed development and germination phases were 59.7%-62.2% and 60.6%-62.8% in Steptoe and Morex, respectively.2. The methylation polymorphisms indicated that about 20% of methylation sites in Steptoe and Morex were changed during seed development and germination. DNA methylation change profiles in two cultivars showed that both methylations and demethylations occurred and demethylation seems to be predominant.3. MSAP analysis demonstrated that relative low level (6.9%) of genetic variation between Steptoe and Morex, whereas about 15% methylation polymorphism between Steptoe and Morex was examined. The epigenetic variations detected by the methylation-sensitive polymorphism were higher than the genetic variations detected by the methylation-insensitive polymorphism. To assay the relationships of methylation variations among different developmental stages of barley seeds, phylogenetic tree was established by hierarchical clustering based on MSAP data. A large divergence in methylation variations were observed between seed germination and maturation process and also between Steptoe and Morex.4. We also cloned and sequenced 36 differential MSAP fragments. The results showed that the lengthes of the analyzed sequences ranged from 70 to 322bp, subsequent blast analysis revealed that cytosine methylated 5'-CCGG- 3'sequences were equally distributed between coding and non-coding regions, and CpG islands which were detected by using Methprimer program could be found in 5 out of the 36 representative sequences of barley.