山核桃甲基化敏感扩增多态体系的建立与甲基化初步分析

山核桃Carya cathayensis为浙江省重要的经济树种。近年来细胞生物学及分子标记的研究发现，山核桃中存在无融合生殖，天然群体的多态性不佳，存在印迹数量性状位点（iQTLs）。iQTLs与DNA甲基化有关，而DNA甲基化对生物表观遗传调控起着重要的作用，是可遗传的生物现象。甲基化敏感扩增多态（MSAP）是基于PCR扩增多态性的DNA甲基化检测方法。在建立山核桃MSAP标记体系的基础上对来自3个天然居群的山核桃单株样品进行了分析。结果表明:山核桃存在甲基化，非甲基化相对水平大于总甲基化相对水平，且两者间差异极显著（Wilcoxon Rank Sum检验P < 0.001），内侧胞嘧啶甲基化相对水平远大于半甲基化相对水平，且两者间差异极显著（分子方差分析及邓肯多重比较，P < 0.001）；天然居群山核桃内侧胞嘧啶甲基化相对水平高，则半甲基化相对水平和非甲基化相对水平则相对较低，反之亦然；居群间存在明显的表观遗传分化，主要变异存在于居群内。此外，山核桃的MSAP体系可用于薄壳山核桃Carya illinoensis，2个物种在位点及位点数上存在差异。

A protocol for methylation-sensitive amplified polymorphism markers and its application to a methylation analysis in Carya cathayensis

In hickory (Carya cathayensis), an important non-timber species in Zhejiang Province, the existence of apomixis, of imprinted quantitative trait loci (iQTLs), and of low DNA polymorphism in terms of cytobiology and molecular markers, has recently been found. Since iQTLs (related to DNA methylation, an inheritable biological phenomenon) played a vital role in epigenetic regulation, methylation-sensitive amplified polymorphism (MSAP), a Polymerase Chain Reaction (PCR)-based method of detecting DNA methylation, was used to establish a protocol in hickory. Then samples from three natural hickory populations were analyzed. Results showed that methylation was prevalent in hickory with the non-methylation level significantly higher (Wilcoxon signed-rank test, P < 0.001) than the total methylation level. Internal cytosine methylation had a relatively higher level than semi-methylation, both of which were also significantly different from each other (Analysis of molecular variance (AMOVA) followed by Duncan's multiple comparison, P < 0.001). If hickory populations had a high level of internal cytosine methylation, then levels of semi-methylation and non-methylation of these populations were relatively low, and vice versa. Also, there was epigenetic differentiation among hickory populations, and the main differentiation existed within a population. In conclusion, this work has laid a foundation for further study of methylation in hickory; in fact, the MSAP protocol established in hickory could be used for pecan, even though the loci and the number of loci for pecan were different from those of hickory.