东乡野生稻耐冷渐渗系DNA甲基化水平和模式变化研究

DNA Methylation Level and Pattern Changes in Cold Tolerance Introgression Lines Derived fromOryza rufipogon Griff.

根据水稻全基因组和特定位点的CpG岛序列设计引物,采用McrBC酶酶切DNA,以东乡野生稻耐冷渐渗系（IL5335和IL5423）及其双亲为试材,研究其基因组和特定位点的DNA甲基化水平和模式变化特征,探讨甲基化变异对野生优异基因渐渗的影响。结果显示：（1）在覆盖全基因组的83个CpG岛中,IL5335和IL5423的基因组甲基化频率分别为46.6%和53.8%,低于受体亲本协青早B的62.6%;大部分（75.9%-80.7%）受体亲本的甲基化模式在两耐冷渐渗中能稳定遗传,另外一些位点发生了甲基化模式的改变,主要表现为脱甲基化（13.3%-18.1%）和过甲基化（4.4%-6.0%）。（2）在耐冷QTLs区间,两耐冷渐渗系的甲基化水平为13.3%-26.7%,远低于受体亲本的61.4%;它们在该区域的甲基化模式变异主要为脱甲基化（33.3%-40.0%）,高于全基因组的平均变异率。（3）分析逆转座子Houba和Osr14区域的51个CpG岛发现,耐冷渐渗系在该区域具有较高频率的甲基化修饰和较低的甲基化模式变异。研究表明,种间杂交渐渗诱发了受体亲本广泛的甲基化水平和模式变异,为野生优异基因的有效利用提供了新的机遇和挑战。

Abstract：In the present study,DNA methylation specific PCR primers were designed according to the se- quences of CpG island region throughout 12 chromosomes and the specific genomic position. In order to in- vestigate the changes of DNA methylation level and pattern in the cold tolerance introgression lines （ILs, IL5335 and IL5423） and their parents,we used these special primers to ampli[y the previously digested ge- nomic DNA by McrBC enzyme. （1）Analysis of 83 pairs of primers distributed across the 12 chromosomes of rice showed that the frequency of DNA methylation in IL5335 and IL5423 was to 46.6% and 53.8%//00 ,re- spectively,which was lower than that of recurrent parent （Oryza sativa L. cv. Xieqingzao B）. Most of DNA methylation patterns of recurrent parent could be inherited in the ILs. At the same time,some of methylation patterns have changed including demethylation （13. 3- 18. 1%） and hypermethylation （4. 4%-6.0%）. （2）In the QTLs region related to cold tolerance,the frequency of DNA methylation in IL5335 and IL5423 was 13.3G-26.7% ,which was much lower than that of recurrent parent. The frequency of DNA methylation patterns changes of the two ILs in the QTLs region was mainly for demethylation （33.3%-40.0%） ,which was higher than that of the average variation frequency of whole-genome. （3）Further analysis on 51 pairs of primers based on two retrotransposons （Houba and Osr14） demonstrated that there was higher level of frequency of DNA methylati0n and lower frequency of methylation pattern changes. These results suggested that the extensive DNA methylation level and pattern changes have been happened during the interspecific hybridization and alien gene introgression, which would provide useful information for efficiently exploiting and using the interesting wild genes for rice enhancement.