DNA Methylation Mechanism And Systematics Of Developmental EFRO In Ginkgo Biloba Var. Epiphylla Mak.

Ginkgo biloba L. had many original and evolutional features. In fact, ginkgo genera maybe was the oldest genera. Numerous researches at home and abroad showed that ginkgo was one of the ideal materials for comparative morphology, anatomy, embryonic development, and phylogenies.As a peculiar germplasm of ginkgo, Ginkgo biloba var. epiphylla Mak, had important value in theory and view. Its distinguishing feature was the ontogeny and structure of epiphyllous female reproductive organ (EFRO), which had important significance in phylogenies, origin, development and research on genetic relationship of ginkgo. Studies on DNA methylation mechanism in EFRO development of Ginkgo biloba var. epiphylla Mak had important significance in revealing morphology essence and systematics. The results and main conclusions as follows:MSAP was applied on ginkgo biloba L., ginkgo biloba var. epiphylla Mak., Cycas, and Adiantum edgeworthii Hook. 16-24 pairs of clear and reproducible selective primers were selected from 54 pairs of MSAP. Cytosine methylation modified level and MSAP amplification patterns of this four species obtained from amplification. The results showed that: about 48.7% (34% full methylation, and 14.7% half- methylation) of the CCGG sites in epiphylla ginkgo genome were methylated, 44.0% (32.8% methylation, and 11.2% half- methylation) in ginkgo, 39.6% (29.2% methylation, and 9.8% half- methylation) in Cycas, and 44.6% (29.4% methylation, and 15.2% half-methylation) in Adiantum edgeworthii. Compared with related species, the methylate level of these species was high. The difference of DNA methylate modified detection rate can reflect the differences in genome species.Cytosine methylation of the CCGG sites at budding stage and leaf-expansion stage of ginkgo and epiphylla ginkgo was general analysis. The results obtained were: at budding stage, methylate level was 47.6% of epiphylla ginkgo, and 42.4% of ginkgo; at leaf-expansion stage, methylate level was 48.3% of malformation-leaf (YZ), 40.5% of normal leaf (YC) of epiphylla ginkgo, and 41.5% of ginkgo leaf (CK), which showed that methylate level of YZ was higher than YC and CK. At the same time, Epiphylla ginkgo and ginkgo had rich variation in methylate model. The ratio of demethylate epiphylla ginkgo to ginkgo at budding sta ge was 13.2%, supermethylation rate was 14.6%, which was little higher than demethylate. The ratio of demethylate YZ to YC at leaf-expansion stage was 8.2%, supermethylation rate was 28%. On the whole,it was obvious that YZ showed supermethylation was highest at leaf-expansion stage,meanwhile, which was also higher than budding stage. Methylate modified level was different at budding stage and leaf-expansion stage of epiphylla ginkgo. It was showed that Cytosine methylation of EFRO of epiphylla ginkgo had specificity with time and space.The cytosine methylate level, mode, and variation type of DNA in different tissue of epiphylla ginkgo was analyzed. It showed that cytosine methylate level DNA in different tissue was different, which was 47.7% at budding stage, at early, middle, late stage of female gametophyte development was 46.0%, 45.2%, and 41.8%, respectively, 35.3% at endosperm stage of CK. Methylate mode was different in different tissue, which showed that demethylate ratio was 19.7%, supermethylation ratio was 14.6% at previous stage, demethylate ratio was 20.0%, supermethylation ratio was 13.3% at middle stage, and demethylate ratio was 20.3%, supermethylation ratio was 14.6% at later stage. The demethylate ratio of CK was 20.3%, and supermethylation ratio was 18.8%. The methylate model difference among different tissue showed that demethylate level was higher than supermethylation level of tissue at budding stage to tissue of three stage of gametophyte development (previous stage, middle stage, and later stage), and early tissue of endosperm of CK. Much gene express at this stage, which was the critical stage of epiphylla ovule formation. This demethylate/supermethylation mode of different stage and different tissue of epiphylla ginkgo laid a foundation for EFRO formation mechanism of epiphylla ginkgo and its phylogenies.16 pairs of selective primers were selected from 54 pairs of MSAP. MSAP amplification pattern produced from different individual of epiphylla ginkgo. Methylate level was different in different individual: youfang (I youfang) was 47.2%, south of zhinv cave (I zhinan) was 44.09%, north of zhinv cave (I zhibei) was 43.8%, baiyu(biayu) was 43.3%, zhonghe (I zhonghe) 43.5%, normal ginkgo of laojuntang (CK) was 39.7%. The methylate mode was also different in individual. Demethylate ratio of I youfang to I zhinan, I zhibei, and CK was 8.7%, 13.3%, and 8.42%, supermethylation ratio of which was 27.1%, 11.22%, and 13.68%, respectively. I youfang appeared higher supermethylation level than other individual contemporaneity, perhaps, which was related with old age (I youfang was 1300a, and others were about 700a). There was relationship between plant morphosis and age. Mode sites of epiphylla ginkgo and ginkgo, and polymorphic site of epiphylla ginkgo were cloned and sequence analyzed by protocols in molecular biology. DNA methylate modified was existed many DNA sequence types, such as: repetitive sequence, transposon sequence, transcription regulator, retrotransposons sequence, F-box protein, channel protein of chloroplast, channel protein of Na+, ubiquitin-protein, hypothetical protein and ethyl-dehydrogenase in epiphylla ginkgo and ginkgo genome by matching homologous sequence. The different in methylate modified level of such genes in epiphylla ginkgo and ginkgo showed that it was helpful to epiphylla ginkgo and ginkgo phenotypic differences formation, and had significance in understanding epiphylla ginkgo formation.