高通量SNP芯片在牛体外早期胚胎染色体质量鉴定中的初步应用

doi:10.11843/j.issn.0366-6964.2022.11.013

摘要/Abstract

摘要： 旨在基于高通量SNP芯片建立一种可评估牛早期胚胎染色体质量和生产性能的方法，为胚胎牛育种提供技术支撑。本研究通过胚胎切割技术分别对荷斯坦奶牛体内囊胚（n=27）、体外囊胚（n=21）、体外2细胞胚胎（n=6）和8细胞胚胎（n=5）进行切割取样并统计发育率，其中体内囊胚组分为1/2体内胚组（切取半个胚胎）和体内滋养层（trophoblast cells，TE）组（切取体内胚胎少量TE），体外囊胚组、体外2和8细胞胚胎分别为体外TE组（切取体外胚胎少量TE）、体外2细胞（切取体外2细胞胚胎的1个卵裂球）和8细胞组（切取体外8细胞胚胎的1个卵裂球）。切割取样后进行全基因组扩增（whole-genome amplification，WGA），对扩增成功且DNA量大于1 000 ng的样品进行SNP芯片检测，芯片检出率大于90%的进行生产性能评估，低于90%的进行染色体片段缺失分析和数据填充。结果显示：1）切割部分TE后，体内TE组剩余部分和体外TE组剩余部分继续培养24 h后的发育率分别为（94.4±5.6）%和（90.5±6.6）%，而1/2体内胚组剩余部分的发育率显著降低，仅为（22.2±14.7）%。2）1/2体内胚组和体外2细胞组扩增成功率为100%，而体内TE组、体外TE组和体外8细胞组扩增的成功率分别为（94.4±5.6）%、（76.2±9.5）%和（60.0±24.5）%；与1/2体内胚组相比，体内TE组和体外TE组经WGA后DNA量均显著下降（P < 0.05），且体外TE组扩增后DNA量显著低于体内TE组（P<0.05）。3）相较于1/2体内胚组（91.2±1.6）%，体内TE组（76.7±15.2）%、体外TE组（74.3±9.6）%、体外2细胞组（76.1±6.9）%、体外8细胞组（61.2±19.0）%芯片检出率均显著下降（P<0.05），且检出率均低于90%。4）以至少连续7个SNPs缺失作为染色体片段缺失筛选标准，体外TE组和体内TE组分别筛选到188个和388个染色体缺失片段，相应的缺失片段各包括46和48个基因；GO和KEGG富集分析结果显示，体外TE组缺失基因主要与细胞骨架和细胞分化相关，而体内TE组缺失基因则主要与细胞物质分泌和运输相关。5）在64 958个SNPs填充位点中52 334个SNPs填充位点的填充准确性（R²）在0.99～1之间且填充准确性为91%，说明填充的准确率较高，育种值估计显示，体外TE组生产性能低于体内TE组。综上，利用胚胎切割、单细胞基因组扩增和基因组芯片可在微创的前提下，实现对植入前早期胚胎染色体质量和生产性能的评估。同时，体内外胚胎染色体缺失片段对比分析发现体外发育过程中细胞骨架等基因异常表达可能是导致体外胚胎质量差的原因之一。

关键词: 牛胚胎, 胚胎切割, 单细胞扩增, SNP芯片, 胚胎质量

Abstract: This study aimed to establish a method that can assess the chromosomal quality and production performance of early bovine embryos, and provide technical support for the industrial application of bovine in vitro embryos. In this study, 27 in vivo blastocyst, 21 in vitro blastocyst, 6 in vitro 2-cell embryos and 5 in vitro 8-cell embryos were selected, and the embryonic cell samples with different cell numbers of in vivo embryos and in vitro embryos were obtained by embryo splitting technique. Among that, in vivo embryos were divided into two groups, one was in vivo trophectoderm cells (tec) group (splitting some of the tec from the in vivo blastocyst), the other was the half in vivo embryo group (splitting half of the embryo from the in vivo blastocyst). The remaining groups were in vitro tec group (splitting some of the tec from the in vitro blastocysts), in vitro 2-cell embryo group (splitting one blastomere from the in vitro 2-cell embryo), and in vitro 8-cell embryo group (splitting one blastomere from the in vitro 8-cell embryo). After embryo splitting, whole-genome amplification (WGA) was conducted, and the samples with successful amplification (DNA amounts greater than 1 000 ng) were subjected to SNP chip detection. Data with a call rate greater than 90% were evaluated for production performance, those below 90% were subjected to chromosomal fragment deletion analysis and data filling. The results showed that:1) After splitting part of the trophectoderm cells, the developmental rates of bovine in vivo embryos (remaining portion of in vivo blastocysts after splitting some of the tec) and bovine in vitro embryos (remaining portion of in vitro blastocysts after splitting some of the tec) were (94.4±5.6)% and (90.5±6.6)%. However, the culture development rate of half of the in vivo embryo (the remaining part of the embryo after splitting half of the embryo from the in vivo blastocyst) was significantly reduced to (22.2±14.7)%. 2) The success rate of DNA amplification in the half in vivo embryo group and the in vitro 2-cell group were 100%, while the success rates of DNA amplification in the in vivo TE group, the in vitro TE group and the in vitro 8-cell embryo group were (94.4±5.6)%, (76.2±9.5)% and (60.0±24.5)%, respectively. Compared with the half in vivo embryo group, the amount of DNA after WGA was significantly lower in both of in vivo TE group and in vitro TE group (P<0.05), and the amount of DNA after WGA was significantly lower in the in vitro TE group than that in the in vivo TE group (P<0.05). 3) Compared to the half vivo embryo group (91.2±1.6)%, the chip call rates were significantly lower in the in vivo TE group (76.7±15.2)%, in vitro TE group (74.3±9.6)%, in vitro 2-cell group (76.1±6.9)%, and in vitro 8-cell group (61.2±19.0)% (P<0.05), and the call rates were all lower than 90%. The in vitro 8-cell group had the lowest chip call rate compared to the other groups. 4) If at least 7 consecutive SNPs deletions were considered as the chromosome fragment deletion selection criteria, a total of 188 and 388 chromosome deletion fragments were obtained in the in vitro TE group and in vivo TE group, respectively. Gene selection identified that a total of 46 genes were included in the deletion fragments of the in vitro TE group and 48 genes were included in the deletion fragments of the in vivo TE group. GO and KEGG enrichment analysis showed that the differentially deleted genes in the in vitro TE group were significantly enriched to cytoskeleton and cell differentiation, whereas the genes enriched in the in vivo TE group were mainly related to cellular material secretion and transport. 5) The filling accuracy (R²) of 52 334 SNPs filling sites out of 64 958 SNPs filling sites was between 0.99 and 1, and the mean value of R² for all filling sites was 91%, indicating a high filling accuracy. Breeding value estimation showed that the production performance of in vitro TE group was lower than that of in vivo TE group. Together, using of embryo splitting, WGA and SNP chips can enable to assess the chromosome quality and production performance of early preimplantation embryos with little impact on the quality of embryo development;In addition, the abnormal expression of genes such as cytoskeleton during in vitro development may contribute to the poor quality of in vitro embryos.

Key words: bovine embryos, embryo splitting, single-cell amplification, SNP chips, embryo quality

中图分类号:

S823.3

胡智辉, 王欢, 衡诺, 巩建飞, 王忆, 王雅春, 赵善江, 朱化彬. 高通量SNP芯片在牛体外早期胚胎染色体质量鉴定中的初步应用[J]. 畜牧兽医学报, 2022, 53(11): 3866-3879.

HU Zhihui, WANG Huan, HENG Nuo, GONG Jianfei, WANG Yi, WANG Yachun, ZHAO Shanjiang, ZHU Huabin. Preliminary Application of High Throughput SNP Chip in Chromosome Quality Identification of Bovine Early in vitro Embryos[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 3866-3879.

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