Estimation of Heterografting Associated DNA Methylation Changes in Tree Crops by MSAP Analysis.

The genetic incompatibility of the seedlings which are used as rootstocks (stock-scion interactions) and the mechanical stress induced by grafting are two major factors responsible for the high intraclonal variations observed in tree crops which are propagated through bud grafting. Since stress-induced DNA methylation changes associated with heterografting is a major contributor of such variations in grafted tree crops, a proper assessment of this epigenetic phenomenon is inevitable to devise strategies for the development of more uniform planting materials with minimal intraclonal variations in the future. In order to evaluate and establish the effects of heterografting on the epigenome of plants, availability of ideal plant materials and a standard procedure for testing is very essential. Development of genetically uniform own-rooted seedlings through induction of cleavage polyembryony by a novel technique of half ovulo embryo culture is the first step. Grafting of buds from these genetically and epigenetically uniform plants to genetically divergent rootstock and identification of DNA methylation polymorphism among them forms the second part of the methodology for detecting epigenetic changes associated with grafting in tree crops. Methylation-sensitive amplification polymorphism technique (MSAP), a modified version of AFLP using a pair of methylation-sensitive and insensitive isoschizomers (such as HpaII and MspI), is an ideal methodology to assess DNA methylation polymorphisms on a genomic scale in such plants. Comparative analysis of two sets of restriction digestion products (EcoRI/HpaII and EcoRI/MspI) allows the identification of DNA methylation polymorphisms induced by grafting and will aid in the detection of differentially methylated regions (DMRs) among grafted plants. This chapter describes a detailed protocol for inducing multiple embryos of single zygotic origin and regeneration of seedlings in rubber tree (Hevea brasiliensis), grafting of buds from these genetically uniform own-rooted seedlings to divergent rootstocks, identification of epigenetic changes induced by grafting or stock-scion interactions through MSAP analysis, and locating the differentially methylated genomic region. The methodology described here could be applied to any tree species commercially propagated through grafting for detecting epigenetic changes putatively associated with intraclonal variability.