基因组甲基化修饰受环境因素的影响。在以甲基化为代表的表观遗传学研究中,如何减少保存环境对异地采后样品的影响,提高整个实验的准确性和科学性,目前尚未有系统的认知。该研究选取5种常用的采后样品保存方式(液氮冷冻、－20℃冷冻、变色硅胶干燥、密封袋密封、75％酒精浸泡),分别用 Wilcoxon signed ranks tests统计分析和UPGMA聚类分析方法,对华南植物园锥栗进行F￣MSAP 研究,以期找出最佳保存方式。同时,利用正交试验法对F￣MSAP 体系进行优化,筛选出9对引物( E3￣H/M2；E5￣H/M2；E6￣H/M1；E6￣H/M5；E8￣H/M1；E8￣H/M5；E9￣H/M2；E11￣H/M5；E14￣H/M1),并对不同发育时期的锥栗甲基化水平及遗传多样性进行了论述。结果表明：在锥栗F￣MSAP 的研究中,Willcoxon signed ranks tests统计分析和UPGMA聚类分析结论一致,密封袋保存效果最佳；成熟叶半甲基化率(27.83％)和总甲基化率(51.13％)高于幼叶(21.35％,45.90％),全甲基化率(23.30％)低于幼叶(24.55％),平均多态位点百分数39.60％,香农信息指数0.207±0.002,表现出较高的甲基化水平和遗传多样性。%Genomic methylation characteristics were influenced by environmental factors. In the study of epigenetics, represented by DNA methylation, the preservation environmental of postharvest samples, especially the samples in the remote place, would have an influence on the later experiment. In this reason, standard sampling and preserve opera￣tion were crucial in the field of epigenetic. But the related research was few. Therefore, we studied Castanopsis chinen￣sis in South China Botanical Garden with F￣MSAP sampling strategy using wilcoxon signed rank tests and unweighted pair￣group method with arithmetic means( UPGMA) methods, in order to find out the best way of sample preservation in five different environments ( Liquid nitrogen, -20℃, allochroic silicagel, hermetic bag and 75% alcohol) . Using the orthogonal experiment method, we optimized the F￣MSAP system and screened nine pairs of primers ( E3￣H/M2,E5￣H/M2, E6￣H/M1, E6￣H/M5, E8￣H/M1, E8￣H/M5, E9￣H/M2, E11￣H/M5 and E14￣H/M1) . Additionally, we briefly described different periods of methylation level and genetic diversity. The results showed that hermetic bag pres￣ervation was the most appropriate way. And the half and total methylation rate of mature leaves was 27. 83% and 51.13%, higher than that of young leaves. The full methylation rate of mature leaves was 23.30%, lower than that of young leaves. The average percentage of polymorphic loci was 39.60%. Shannon information index was 0.207 ± 0.002, which showed a higher level of methylation and genetic diversity.
展开▼
