2.3. Quantifying Variation

Estimation of TCIV was based on the presence (1) or absence (0) of bands (DNA fragments) for donors and regenerants on a polyacrylamide gel. Binary matrices were created based on the compilation of DNA profiles obtained from Acc65I/MseI and KpnI/MseI enzyme cuts. Based on the properties of the restriction enzymes used, it was possible to determine the changes in the DNA of regenerants caused by in vitro culture. Sequence variation (SV), demethylation (DMV), and de novo methylation (DNMV) were determined. Selective primers terminated with any combination of A and T could amplify the asymmetric CHH sequence. MetAFLP primers terminated with the -CHG sequence at the 3′-end and those with the -CG sequence reflect symmetric contexts. Thus, selective primers targeting symmetric and asymmetric DNA sequences used in the metAFLP technique enabled identification of CG, CHG, and CHH methylation change contexts as well as sequence changes related to those sequences. The details of quantifying changes in different sequence contexts concerning DNMV, DMV and SV were described elsewhere [33].