太平洋牡蛎不同组织DNA甲基化的F-MSAP分析

Analysis of DNA methylation in different tissues of the Pacific oyster(Crassostrea gigas) with the fluorescence-labeled methylation-sensitive amplified polymorphism(F-MSAP)

采用荧光标记的甲基化敏感性扩增多态性(F-MSAP)技术对太平洋牡蛎(Crassostrea gigas)的6个组织进行全基因组CCGG位点甲基化水平的检测。结果表明:鳃、闭壳肌、外套膜、唇瓣、性腺、消化盲囊6个组织的甲基化水平分别为:31.77%、36.41%、32.00%、36.47%、32.77%和37.92%。甲基化模式分为全甲基化和半甲基化,在太平洋牡蛎中,全甲基化模式与总体甲基化水平一致,消化盲囊、唇瓣、闭壳肌组织的甲基化水平较高,而鳃、外套膜、性腺组织的甲基化程度较低;半甲基化位点显著少于全甲基化位点,并且不同组织间的半甲基化位点不存在显著性差异,而从全甲基化位点及总甲基化水平分析,不同组织之间甲基化水平差异显著。太平洋牡蛎中存在组织特异性甲基化片段和非组织特异性甲基化片段,但却只在一个或部分个体中出现,没有发现在所有研究个体中都存在的甲基化差异片段,这可能与牡蛎较高的杂合度相关。对实验样品进行的荧光标记的扩增片段长度多态性(F-AFLP)分析发现,样品间的确存在较大的遗传差异,基于以上结果可以推测基因组甲基化水平可能与个体遗传背景相关联。

The DNA methylaiton extent and patterns of CCGG sites in the genome of the Pacific oyster （Crassostrea gigas） was investigated among six different tissues using the fluorescence-labeled methyla-tion-sensitive amplified polymorphism （F-MSAP） method. The incidence of DNA methylaiton in the six tissues （gill, adductor, mantle, labial palp, gonad and digestive glands） was 31.77%, 36.41%, 32.00%, 36.47%, 32.77%and 37.92%, respectively. DNA methylation patterns can be divided into two types： the fully methylated and hemimethylated loci. The fully methylated loci showed similar trend with the total methylation level： digestive glands, labial palp and adductor showed much higher methylation level than the other three tissues and the differ-ence between the six tissues was significant （P〈0.05）. The rate of hemimethylated loci is less than the fully me-thylated loci and not significantly different between the tissues. Both tissue specific （bands methylated or un-methylated in only one of the six tissues） and tissue nonspecific differentially methylated regions （bands methy-lated or unmethylated in more than one of the six tissues） were found in Pacific oyster, but these two kinds of bands only occurred in one or few individuals. No tissue specific/nonspecific differentially methylated regions were found in all individuals analyzed and this might correlate with the high polymorphism in the Pacific oyster. Fluorescence-labeled amplified fragment length polymorphism （F-AFLP） showed the percentage of polymorphism loci was 87.5%, and the expected heterozygosity ranged from 0.267 0 to 0.347 5. Results from F-MSAP and F-AFLP indicated that genetic variation might contribute to the DNA methylation patterns.