Genome-wide and single-base resolution DNA methylomes of the Pacific oyster Crassostrea gigas provide insight into the evolution of invertebrate CpG methylation

Comparative analysis of the oyster DNA methylomes and that of other animal species revealed that the characteristics of DNA methylation were generally conserved during invertebrate evolution, while some unique features were derived in the insect lineage. The preference of methylation modification on genes originating in the eukaryotic ancestor rather than the oldest genes is unexpected, probably implying that the emergence of methylation regulation in these 'relatively young' genes was critical for the origin and radiation of eukaryotes. Two Pacific oysters were used for methylome profiling in this study. One is an inbred oyster (05x7-T-G4-1.051#20) that was produced by four generations of sister-brother mating (coefficient of inbreeding, F = 0.59) and has been used for whole genome-sequencing. The other was a wild oyster collected from Weihai, Shandong Province, China. Both oysters were about two years of age. The inbred oyster was produced as single oysters and cultured intertidally at southern Puget Sound, Washington, USA, where the water temperature ranges from 7 to 16 °C. The wild oyster was an attached oyster from an oyster farm in its native range at Weihai, China, where the water temperature ranges from 4 to 27 °C. For DNA from each of the two individuals, we constructed two independent libraries. For each library, 5 µg genomic DNA mixed with 25 ng cl857 Sam7 Lambda DNA was fragmented by sonication with a Covaris S2 system (Covaris, MA) to a mean size of approximately 250 bp. End-repair, 3’-end dA addition and adapter ligation were subsequently performed. Methylated adapters were used according to the manufacturer’s instructions (Illumina). The bisulfite conversion of DNA was performed according to a modified NH4HSO3-based protocol and amplified with nine cycles of PCR. All libraries were subjected to 90-bp paired-end sequencing on an Illumina HiSeq 2000 platform.

本研究通过对太平洋牡蛎（Pacific oyster）与其他动物物种的DNA甲基化组（DNA methylome）开展比较分析，结果显示DNA甲基化的特征在无脊椎动物演化过程中总体保持保守，而昆虫谱系则演化出部分独特特征。甲基化修饰偏好作用于真核生物祖先起源的基因而非最古老的基因，这一发现出人意料，或暗示在这些"相对年轻"的基因中出现甲基化调控，对真核生物的起源与辐射演化至关重要。本研究共选取两只太平洋牡蛎进行DNA甲基化组谱分析：其中一只为近交系牡蛎（05x7-T-G4-1.051#20），该品系经四代兄妹交配培育而成（近交系数F=0.59），此前已用于全基因组测序；另一只为采自中国山东省威海市的野生牡蛎。两只牡蛎均为约2龄个体。该近交系牡蛎为单只个体，于美国华盛顿州普吉特湾南部潮间带养殖，该海域水温范围为7至16℃；野生牡蛎为采自中国威海原产地牡蛎养殖场的附着型牡蛎，该养殖海域水温范围为4至27℃。针对两只个体的基因组DNA，我们分别构建了两个独立的测序文库。每个文库均使用5μg基因组DNA，混合25 ng cl857 Sam7 Lambda DNA，通过Covaris S2超声破碎仪（Covaris，马萨诸塞州）将DNA片段化至平均长度约250 bp。随后依次完成末端修复、3’端加dA尾以及接头连接操作。甲基化修饰接头的使用严格遵循Illumina官方说明书进行。DNA的亚硫酸氢盐转化参照基于NH4HSO3的改良方案实施，并通过9轮PCR进行扩增。所有文库均在Illumina HiSeq 2000测序平台上完成90 bp双端测序。