Genotipizzazione di linee mutanti per epiregolatori in mais

In the last years, it has been proved that epigenetic modifications such as DNA methylation, production of sRNAs, modification of histone tails and the presence of histone variants may act to alter the level of chromatin condensation, regulating gene expression in spatially and temporally way. These epigenetic mechanisms act together to fine regulate gene expression in plants and mammals, especially in response to environmental factors. Studing the mechanisms and the processes that lead to the formation and propagation of epialleles in maize is the subject of the research project in which this thesis is inserted and of this thesis as well. In particular we are interested in understanding how environmental stresses can alter epigenetic regulation of gene expression, leading to the formation of stable, inheritable epialleles. The final goal of the project would be the use of this stress-induced epigenetic variability in new breeding program in maize. To do this we are producing mutants for different epigenetic regulators (epi-regulators) and we are setting up different stress protocols in maize to identify new epitargets, that are genes induced by stress and regulated epigenetically. Insertional mutant lines used were obtained by crossing a wild-type maize line with pollen derived from a line in which the Mu transposon resulting in a hybrid background. It is therefore necessary to backcross this mutant lines with the B73 wild type inbred line several times (at least five) to introgress the mutations in this reference background. B73 in indeed the maize inbred line for which the genome has been completely sequenced, and only after the completion of the introgression the mutant plants will be selfed twice to obtain homozygous mutants. The first part of the thesis work, plants of 5 mutants lines for different epiregulators were genotyped, proceeding to the DNA extraction and to the application of PCR (Polymerase Chain Reaction) to identify the plants that carry the insertional mutation. To perform the PCR reaction a universal primer, designed on the sequences terminal inverted and repeat (TIR) of the transposon, was used in combination with one or more gene-specific primers, allowing amplification of the fragment of interest only from mutant plants. The so indentified plants have been then crossed to B73 plants, getting along the backcross program.