牛活检胚胎细胞微量扩增体系效果评估

doi:10.11843/j.issn.0366-6964.2025.01.023

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (1): 246-258.doi: 10.11843/j.issn.0366-6964.2025.01.023

牛活检胚胎细胞微量扩增体系效果评估

牛一凡¹^,²(), 李崇阳¹, 张培培¹, 张航¹, 冯肖艺¹, 余洲¹, 曹建华¹, 杜卫华¹, 万鹏程³, 马友记²^,*(), 赵学明¹^,*()

1. 中国农业科学院北京畜牧兽医研究所, 北京 100193
2. 甘肃农业大学动物科学技术学院, 兰州 730070
3. 新疆农垦科学院畜牧兽医研究所, 石河子 832000

收稿日期:2024-07-02 出版日期:2025-01-23 发布日期:2025-01-18
通讯作者: 马友记,赵学明 E-mail:nyf.niuyifan@qq.com;yjma@gsau.edu.cn;zhaoxueming@caas.cn
作者简介:牛一凡(1999-)，男，河北邯郸人，硕士生，主要从事动物繁殖研究，E-mail: nyf.niuyifan@qq.com
基金资助:
国家重点研发计划政府间重点专项(2022YFE0100200)；国家自然科学基金国际合作项目(32161143032)；农业农村部和财政部资助：现代农业产业技术体系资助(CARS-36)；国家家养动物种质资源库；中国农业科学院科技创新工程(ASTIP-IAS06)

Microamplification System Evaluation of Bovine Biopsied Embryo Cells

NIU Yifan¹^,²(), LI Chongyang¹, ZHANG Peipei¹, ZHANG Hang¹, FENG Xiaoyi¹, YU Zhou¹, CAO Jianhua¹, DU Weihua¹, WAN Pengcheng³, MA Youji²^,*(), ZHAO Xueming¹^,*()

1. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China
2. College of Animal Science and Technology of Gansu Agricultural University, Lanzhou 730070, China
3. Institute of Animal Husbandry and Veterinary, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China

Received:2024-07-02 Online:2025-01-23 Published:2025-01-18
Contact: MA Youji, ZHAO Xueming E-mail:nyf.niuyifan@qq.com;yjma@gsau.edu.cn;zhaoxueming@caas.cn

摘要/Abstract

摘要：

旨在通过二代测序技术评估不同单细胞全基因组扩增体系对不同数量胚胎细胞的扩增效率。本研究选择同一饲养管理条件下、体况良好的华西牛母牛为供体，采用FSH法超数排卵后进行人工授收集第7天胚胎，通过活检技术获得含1细胞(cell，1C)、5C、10C胚胎细胞样本用于全基因组扩增(whole genome amplification)，随后通过全基因组重测序(whole genome sequencing)技术评估单细胞基因组扩增体系扩增效率的差异。结果显示，在1C和5C样本中多重置换扩增(multiple displacement amplification，MDA)体系的扩增成功率优于多次退火环状循环扩增(multiple annealing and looping-based amplification cycles，MALBAC)体系(100% vs. 60%，100% vs. 80%，P < 0.05)，此外MDA体系的扩增产物总量极显著高于MALBAC体系(P < 0.01)；基于测序分析指标：正确比对率、重复率、分型一致率、等位基因缺失率、假阳性率等，MALBAC体系优于MDA体系；全同胞样品的分型一致率大于半同胞样品的分型一致率，但不同全同胞、半同胞胚胎样本之间的分型一致率有很大差异。综上所述，MDA和MALBAC体系各具特点，结合两者优势可能是改进扩增效率的途径之一，本研究为牛早期胚胎全基因组选择研究和促进牛育种发展提供一定理论基础。

关键词: 华西牛, 活检胚胎, 扩增体系, 效果比较

Abstract:

The purpose of this study was to evaluate the amplification efficiency of different single cell whole genome amplification systems for different numbers of embryonic cells by second generation sequencing technology. In this study, healthy Huaxi cows under the same feeding and management conditions were selected as donors, and the 7th day embryos were collected after superovulation by FSH method. Samples containing 1C(cell), 5C and 10C embryos were obtained by biopsy technique for whole genome amplification, and then the amplification efficiency of single cell genome amplification system was evaluated by whole genome resequencing techniques. The results showed that the amplification success rate of multiple displacement amplification (MDA) system was better than that of the multiple annealing and looping-based amplification cycles (MALBAC) system in 1C and 5C samples (100% vs. 60%, 100% vs. 80%, P < 0.05). In addition, the total amount of amplification products of MDA system was significantly higher than that of MALBAC system (P < 0.01). Based on the sequencing analysis index: correct mapping rate, dup rate, typing consistency rate, allele drop-out rate and false positive rate, etc., MALBAC system was better than MDA system. The typing consistency rate of full-sib samples was higher than that of half-sib samples, but there were great differences between different full-sib and half-sib embryo samples. To sum up, MDA and MALBAC systems have their own characteristics, and the combination of their advantages may be a way to improve amplification efficiency. This study provides a theoretical basis for the whole genome selection of early bovine embryos and promoting the development of bovine breeding.

Key words: Huaxi cattle, embryo biopsy, amplification system, effect comparison

中图分类号:

S823.3

牛一凡, 李崇阳, 张培培, 张航, 冯肖艺, 余洲, 曹建华, 杜卫华, 万鹏程, 马友记, 赵学明. 牛活检胚胎细胞微量扩增体系效果评估[J]. 畜牧兽医学报, 2025, 56(1): 246-258.

NIU Yifan, LI Chongyang, ZHANG Peipei, ZHANG Hang, FENG Xiaoyi, YU Zhou, CAO Jianhua, DU Weihua, WAN Pengcheng, MA Youji, ZHAO Xueming. Microamplification System Evaluation of Bovine Biopsied Embryo Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 246-258.

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体系名称 System	样品数量 Sample quantity	细胞数量 Cell number	原始测序量/G Raw date	原始reads Raw reads	Q20/%	Q30/%	GC/%
NC	1	/	78.08	240 265 726	97.28	92.27	42.77
MDA	4	1C	190.69	635 613 479	98.30	94.34	42.37
	4	5C	225.79	752 633 841	98.30	94.23	42.69
	5	10C	301.29	1 004 318 819	98.22	93.93	40.69
MALBAC	3	1C	159.68	532 248 347	98.27	94.23	43.43
	4	5C	217.24	724 110 484	98.13	93.82	43.22
	6	10C	343.16	1 143 852 069	98.15	93.78	43.19

样本1 Sample1	样本2 Sample2	细胞数量 Cell number	样本关系 Sample relationship	分型一致率/% Consistent rate of typing	分型一致位点数 The number of consistent loci of typing	分型不一致位点数 The number of inconsistent loci of typing
Y1	Y2	1C	全同胞	79.93	15 423 363	3 872 311
Y3	Y4	5C		77.86	14 664 766	4 169 400
Y5	Y6	10C		84.02	16 163 185	3 075 191
Q1	Q2	10C		87.82	23 650 146	3 279 974
Y2	Y4	5C	半同胞	76.38	14 269 464	4 413 134
Y4	Y5	10C		79.28	14 567 703	3 807 469
Y2	Y5	10C		77.88	15 036 581	4 270 454
Q3	Q4	5C		82.28	20 365 863	4 385 250
Q5	Q2	10C		82.25	22 009 254	4 748 595
Q6	Q2	10C		81.84	21 673 120	4 808 018

[1]	牛一凡, 李崇阳, 杨柏高, 张培培, 张航, 冯肖艺, 曹建华, 余洲, 马友记, 赵学明. 不同单细胞全基因组扩增体系扩增牛微量血液DNA效果评价[J]. 畜牧兽医学报, 2024, 55(8): 3436-3445.
[2]	李柯安宁, 杜丽丽, 安炳星, 邓天宇, 梁忙, 曹晟, 杜悦莹, 徐凌洋, 高雪, 张路培, 李俊雅, 高会江. 华西牛胴体及原始分割肉块重量性状遗传参数估计与全基因组关联分析[J]. 畜牧兽医学报, 2023, 54(9): 3664-3676.

牛活检胚胎细胞微量扩增体系效果评估

Microamplification System Evaluation of Bovine Biopsied Embryo Cells

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参考文献 43

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细胞数量 Cell number	体系名称 System name	扩增产物浓度/(ng·μL^-1) Post-amplification concentration	体积/μL Volume	扩增产物总质量/μg Total mass
1C	MDA	754.50±57.28^A	50	37.73±2.86^A
1C	MALBAC	24.17±11.17^B	65	1.57±11.17^B
5C	MDA	941.00±174.37^A	50	47.05±8.72^A
5C	MALBAC	29.35±0.41^B	65	1.91±0.02^B
10C	MDA	805.60±20.27^A	50	40.28±1.01^A
10C	MALBAC	26.87±1.40^B	65	1.75±0.09^B

扩增体系 Amplification system	扩增样品总数 Amplify the total number of samples	扩增成功样品数 The number of samples successfully amplified	扩增成功率/% Amplification success rate
MDA	14	13	92.86
MALBAC	16	13	81.25