桔梗同源四倍体诱导及其基因组DNA甲基化差异分析

In vitro autotetraploid induction and analysis on DNA methylation diversity of Platycodon grandiflorum

为探究桔梗同源四倍体与二倍体试管苗外观形态差异的表观遗传变化,先将桔梗二倍体不定芽用不同浓度的秋水仙素(加0.02 g·m L-1二甲亚砜)混合液浸泡后进行组织培养,并通过流式细胞仪和根尖染色体鉴定相结合的方法鉴定倍性。在获得桔梗同源四倍体的基础上,采用甲基化敏感扩增多态性(methylation sensitive amplified polymorphism,MSAP)技术探究二倍体与四倍体植株之间的DNA甲基化水平及其模式变化。结果表明0.2%的秋水仙素(加0.02 g·m L-1二甲亚砜)混合液处理桔梗不定芽12 h是诱导桔梗同源四倍体的最适条件,其诱导率达到32.0%;四倍体与二倍体植株在外观形态上存在明显差异,经过DNA甲基化差异分析,20对引物扩增的1 586条带中二倍体有764条,四倍体有822条。二倍体总甲基化率、全甲基化率和半甲基化率分别为92.15%,61.52%,30.65%,而四倍体为86.13%,54.38%,31.75%,与二倍体相比,其同源四倍体总甲基化率降低6.02%,全甲基化率降低7.14%,而半甲基化率升高1.6%,说明染色体加倍后其DNA表观遗传模式发生了变化。

In order to investigate the epigenetic variations between diploid and autotetraploid of Platycodon grandiflorus. the diploid buds of P. grandiflorus were soaked in the mixture of different concentration colchicines and 0. 002 g · m L- 1dimethyl sulphoxide（ DMSO）. The identification of autotetraploid plants were based on morphological characteristics,chromosome number and flow cytometry. And then the level and pattern of DNA methylation explored by using the technology of methylation sensitive amplified polymorphism（ MSAP）. The result demonstrated that the buds soaked in 0. 2% colchicines and 0. 002 g·m L- 1DMSO solution for 12 h was ideal conditions to induce autotetraploid of P. grandiflorus,with induction rate of 32. 0%. The diploid and tetraploid plants existed distinctly differences in morphological indexes. Totally,1 586 bands were amplified by 20 pairs of selective primers,of which 764 and 822 bands were detected in diploid and autotetraploid respectively. The total methylation ratio,full methylation ratio and hemimethylated ratio were 91. 25%,61. 25% and 30. 65% in diploid of P. grandiflorus,respectively. However,the total methylation ratio,full methylation ratio and hemimethylated ratio of autotetraploid of P. grandiflorus were 86. 13%,54. 38% and 31. 75%,respectively. Compared with diploid,the genomic DNA total methylate ratio and full methylation ratio of autotetration plants decreased by 6. 02% and 7. 14%. But the hemimethylated ratio of autotetraploid was higher than that of diploid,which more than 1. 6%. All this results indicated that DNA methylation patterns have adjusted during the polyploidy process.