Inheritance And Variation Of DNA Methylation Level And Pattern In Leaf And Endosperm Of Sorghum (Sorghum Bicolor L.)

DNA methylation as an important epigenetic modification plays an important role in controlling gene expression and maintaining genomic stability in animals and plants. Understanding the dynamics of DNA methylation associated with development, and transmission genetics of methylation patterns across organismal generations are important facets for elucidating the roles played by epigenetic paradigms in plant development and genome evolution. Using four sets of sorghum (Sorghum bicolor L.) inter-strain hybrids and their inbred parental lines, we investigated the developmental stability, as well as inheritance and variation of level and pattern of cytosine methylation in two tissues, leaf and endosperm, by the methylation-sensitive amplified polymorphism (MSAP) method. The main results were summeized as following.1. In all sorghum lines (inbred and hybrid) studied, endosperm exhibited a markedly reduced level of full-methylation of the external cytosine or both cytosines at the CCGG sites relative to leaf (endosperm-specific hypomethylation), which caused a variable reduction in the estimated total methylation level in endosperm by 6.89-19.69% (11.47% on average), thus implying a general epigenetic reprogramming during seed development in sorghum.2. For both tissues, a great majority of cytosine methylation profiles transmitted to F1 hybrids, nonetheless, from 3.16% to 8.09% of the profiles showed altered patterns in hybrids relative to their inbred parents, mainly resulting from non-inheritance of certain parental profiles, which contributed to a seemingly reduced methylation level in hybrids compared with the inbred parental lines (particularly in leaf, which is statistically significant). Both inherited and altered methylation profiles can be divided into distinct groups, and their frequencies are variable among the cross-combinations, and between the two tissues.3. Evidence was obtained that the variations in cytosine methylation level and pattern in hybrids were not caused by parental heterozygosity, and they could be either non-random or stochastic among hybrid individuals.4. Homology analysis of isolated bands that showed endosperm-specific hypomethylation or typical variation in hybrids indicated that diverse sequences were involved, including known-function cellular genes, mobile elements and uncharacterized sequences.5. The inheritance and variation of level and pattern in cytosine DNA methylation from inbred parents to hybrids were validiated by Southern blot analysis.6. RT-PCR analysis of six genes representing endosperm-specific hypomethylation indicated that all showed higher expression in endosperm than in leaf, suggesting involvement of methylation state in regulating tissue-specific or biased expression in sorghum. Analysis on leaf-RNA from 5-azacytidine-treated plants further corroborated this possibility.The above findings suggest that endosperm development in sorghum is accompanied by specific hypomethylation, which at least for some genes, is associated with endosperm-specific or enhanced expression. Compared with the parental inbred lines, in both tissues (leaf and endosperm) of a given sorghum hybrid the great majority of loci exhibited additive inheritance, nonetheless, variation occurred at certain loci. It thus appeared plausible that the dynamics of DNA methylation level and pattern in the course of ontogenic development might play a role in regulating gene expression. On the other hand, the variation in level and pattern of DNA methylation in a hybrid sorghum plant might contribute to the non-additive gene expression relative to its parental inbred lines.