Study On The DNA Methylation/Demethylation During Somatic Embryogenesis And Mining Of SSR Markers In Citrus

Citrus was one of important crops in the world and markers assisstant screening was one of important methods for citrus breeding. In this work, two kinds of molecular markers were developed and applied in citrus. The first one was the development and application of ASAP (methylation-sensitive amplification polymorphism) markers. MSAP was a kind of AFLP markers in combinations with DNA methylation sensitive enzymes. DNA methylation that was a kind of modification of DNA had played an important role in the regulation of genes. And regeneration from callus was for citrus one of main approachs of reproduction which had widely ustilized in modern biotechnology. But some key factors which were still unknown impaired the development of the approach. In this study, AFLP-based technology MSAP was used to detect DNA methylation changes during the regeneration of callus, in order to uncover the role of DNA methylation played in the regeneration of citrus callus. The second was the development of microsatellites (also called SSRs) markers which had widely used in citrus. But the limited markers block the futher applications of SSRs in citrus. In this study, we tried to develop SSRs directly from sequence databases. The main results were as follows:1 Investigating plant abnormalities during the regeneration of sweet orange and Hongkong Kumquat (Fortunella hindsii). Both normal and abnormal plants could regenerate from embryoids and abnormality was unstable during the development of plant. It indicated that the abnormality was uneven and reversible which was similar with that induced by DNA methylation in Arabidopsis.2 A Hongkong Kumquat single-embryo-line was established; the callus, embryoids and leaves from the single-embryo-line plus long time subcultured callus were used as samples. 72 primer pairs of PsiI/MseI and HpaII, MspI/EcoRI combinations produced 53, 19 polymorphic bands individually. It indicated that MSAP could assess efficifently DNA methylation changes during the regeneration of callus.3 During the regeneration of citrus, three patterns were found: bands appearance, bands disappearance, bands appearance and then disappearence. It indicated that both methylation and demethylation event were observed during the regeneration of callus. The level of DNA methylation increased during the regeneration and subculture. Southern...