Zygotes segregate entire parental genomes in distinct blastomere lineages causing cleavage stage chimaerism and mixolpoidy

Chimaerism and mixoploidy define the presence of cell lineages with different parental genomes or different ploidy states in a single individual. Our knowledge on their mechanistic origin results from indirect observations, often when the cell lineages have been subject to rigorous selective pressure during development. Here, we applied haplarithmisis to infer the haplotypes and the copy number of parental genomes in 116 single blastomeres comprising entire preimplantation stage bovine embryos (n=23) following in vitro fertilization. Not only abnormal fertilizations leading to triploid zygotes, but also normally fertilized zygotes can spontaneously segregate entire parental genomes into different cell lineages during cleavage of the zygote. We coin the term “heterogoneic division” to indicate the events leading to non-canonical zygotic cytokinesis segregating the parental genomes into distinct lineages. Persistence of those cell lines during development is the likely cause of chimaerism and mixoploidy in mammals. Oocytes from eight Belgian Blue cows (Bos taurus) and semen from two Holstein-Friesian bulls (Bos taurus) were used for embryo production (BRP004 to BRP012 crosses). Eleven embryos were isolated at day-2 post insemination (pi) and 14 embryos were isolated on day-3 pi. Ovarian tissue from the donor cows (mothers) and semen from the two bulls (fathers) were used to extract bulk DNA (DNeasy Blood and Tissue kit, Qiagen). In addition, we obtained bulk DNA from the parents of the bulls (i.e. paternal grandparents of embryos). Moreover, DNA samples extracted from single blastomeres and the two expanded blastocysts (day-8 pi of BRP010 and BRP011 crosses) were whole genome amplified using the REPLI-g Single Cell Kit according to the manufacturer’s protocol (Qiagen). Subsequently, Single-cell and multi-cell parental or sibling DNA genotype calls, BAF and logR values were obtained from the BovineHD BeadChip raw intensity data by application of the GenCall algorithm, which is embedded in Illumina’s GenomeStudio software (http://illumina.com/software/genome_studio_software.ilmn). Genotypes were called by setting the GenCall score at 0.75 (based on the optimization steps described in Zamani Esteki et al. 2015). The raw LogR- and BAF-values as well as discrete SNP genotype calls were fed to a modified version of siCHILD algorithm (Zamani Esteki et al. 2015).

嵌合现象（chimaerism）与混倍性（mixoploidy）指单个个体内存在携带不同亲本基因组或不同倍性状态的细胞谱系的现象。目前学界对其机制起源的认知均来自间接观测，且这类观测通常是在细胞谱系经历发育过程中的严格选择压力后开展的。本研究采用单倍型推算（haplarithmisis）技术，对23枚完整体外受精（in vitro fertilization, IVF）牛植入前胚胎来源的116个单细胞卵裂球的亲本基因组单倍型与拷贝数进行推断。研究发现，不仅异常受精可形成三倍体合子，正常受精的合子在卵裂阶段也可自发将整套亲本基因组分离至不同的细胞谱系中。我们将合子胞质分裂时将亲本基因组分离至不同细胞谱系的非经典事件命名为"异向分裂（heterogoneic division）"。这类细胞谱系在发育过程中的持续存在，可能是哺乳动物发生嵌合现象与混倍性的诱因。本研究以8头比利时蓝牛（Bos taurus）的卵母细胞与2头荷斯坦-弗里生公牛（Bos taurus）的精液为材料，开展胚胎生产（涉及BRP004至BRP012的杂交组合）。分别于输精后（post insemination, pi）第2天分离11枚胚胎，第3天分离14枚胚胎。以供体母牛（胚胎母本）的卵巢组织与两头公牛（胚胎父本）的精液为材料，使用Qiagen的DNeasy血液与组织试剂盒提取批量基因组DNA。此外，我们还从公牛的亲本（即胚胎的父系祖父母）处提取了批量基因组DNA。针对从单个卵裂球以及2枚扩张囊胚（BRP010与BRP011杂交组合输精后第8天的胚胎）中提取的DNA样本，我们按照Qiagen REPLI-g单细胞试剂盒的说明书进行全基因组扩增。随后，依托嵌入Illumina GenomeStudio软件（http://illumina.com/software/genome_studio_software.ilmn）的GenCall算法，从牛HD基因芯片（BovineHD BeadChip）的原始强度数据中获取单细胞与多细胞亲本或同胞的DNA基因型分型结果、等位基因频率（B-allele frequency, BAF）与对数比值（log R ratio, logR）数值。基因型分型的GenCall评分阈值设置为0.75（参照Zamani Esteki等人2015年描述的优化步骤）。将原始logR与BAF数值以及离散的单核苷酸多态性（single nucleotide polymorphism, SNP）基因型分型结果输入至经改造的siCHILD算法（Zamani Esteki等人2015年开发）中。