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

Abstract

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

CLC Number:

S823.3

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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References 43

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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

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Microamplification System Evaluation of Bovine Biopsied Embryo Cells

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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