Effects Of High-voltage Electric Field On DNA Methylation During Seed Germination Of Brassica Oleracea Var.Acephala

Brassica oleracea Var. acephala is a typical cross-pollination plant with remarkable heterosis. Self-incompatible lines with excellent comprehensive characters by self-pollination purification using self-incompatibility of this cultivar were established, and later new varieties were obtained by using the self-incompatible lines as crossing parents. The progeny of this self-incompatible cultivar have strong degradation phenomena by self-pollination of many generations.1000-seed weight of this cultivar was almost half of that of self-compatible line; growth vigor decline and they were susceptible to many diseases. These unfavorable factors affected the improvement of this cultivar seriously, and brought difficulties to cross breeding. Therefore, effective means to retard delay and restore growth vigor, and keep the quality of self-bred progeny was meaningful to propagate self-incompatibility lines and cross-breed them.DNA methylation plays an important role in regulating plant growth, and it is necessary for plant normal growth and development. In this experiment, methylation sensitive amplification polymorphism(MSAP) was used to study the status and patterns of the DNA methylation at different periods in seed germination of self-incompatible line9#and the change of DNA methylation status in9#after high-voltage electric field. In addition, self-incompatible line9#was compared with self-compatible line14#in DNA methylation level at0hour and48hours after seed germination. The corrslation was discussed between seed degradation phenomena of self-incompatible line and epigenetic one. The results are shown as follows:1. DNA methylation modification throughout the whole process of seed germination of9#, DNA methylation levels changed dynamically in seed germination, the CCGG sites of genomic cytosine methylation was given priority to with full methylation. Methylation and demethylation were detected during the whole germination stages; and mainly demethylation was observed. At the early stage of the germination(0～48hours) methylation sites continued to increase; at the later stage (48hours～8days) demethylation increased apparently, and eventually the number of demethylation was11times more than methylation. During germination of9#seeds, DNA methylation patterns were numerous and changed frequently, and sites involved were multitudinous. There were some loci where methylation patterns changed repeatedly; but some sites were repeatedly methylation or demethylation. It was proved that DNA methylation modification was an important way to regulate the gene expression during seed germination of self-incompatible line9#2. self-incompatible line9#and self-compatible line14#had different DNA methylation status clearly at the respectively periods of0hour and48hours after seed germination. The proportion of total methylation, full-methylation and semi-methylation of9#was all higher than of14#at the same period. As seedings continued to grow after germination, in9#the proportion of full-methylation increased clearly and almost that of semi-methylation did not change, while in14#the proportion of semi-methylation increased clearly and almost that of full-methylation did not change.3. The physiological indexs of9#in seed germination,which showed high-voltage electric field, could promote seed germination and seeding growth and improve the quality of seeds. High-voltage electric field was an effective means to delay and restore growth vigor, and to maintain the quality of self-crossed progeny.4kV/cm,30minutes seemed to be the best treatment dose.4.1tried to explain the molecular mechanism of high voltage electric field could promote seed germination and seeding growth from the perspective of epigenetics using MSAP. After high-voltage electric field treatment, the proportion of full-methylation increased slightly and semi-methylation declined clearly and brought about the result of total methylation declined.19.64%of methylation sites were changed in methylation status, both methylation and demethylation occurred; and demethylation seemed to be predominant.5. The differenrial MSAP fragments were recycled and matched by Blastn, and many genes were identified to be closely related with plant growth and development, i.e., signal transduction, growth metabolism, cell organelle structure, transcription factor, and kinase biological processes. The results enriched the diversity of genome methylation sites of Cruciferae. These genes which were related with plant growth and development seemed to be regulated probably by the process of methylation and demethylation directly or indirectly. This study provided theoretical basis for the study of degradation phenomena in self-incompatible line of Brassica oleracea Var. acephala at molecular level.