绵羊(ATAG)n四碱基微卫星位点的筛选及其在亲子鉴定中的应用

doi:10.16431/j.cnki.1671-7236.2021.07.024

摘要/Abstract

摘要： 本试验旨在筛选绵羊高度多态性四碱基微卫星遗传标记，建立适用于绵羊亲子关系鉴定的实验体系。试验以小尾寒羊为主要研究对象，从已有的绵羊参考基因组序列出发，基于全基因组序列筛选法共筛选出53个（ATAG）_n四碱基重复微卫星位点，然后通过基因分型数据共筛选出30个扩增效果好、多态信息含量（PIC）丰富的四碱基重复微卫星位点；30个位点的基因分型结果表明，共扩增出253个等位基因，平均等位基因数为8.433，等位基因数均>5，多态信息含量在0.566~0.898，观测杂合度（Ho）范围在0.548~0.903，期望杂合度（He）范围在0.631~0.921，平均期望杂合度为0.776；哈代-温伯格平衡检验30个位点均处于遗传平衡状态。随后根据PCR的扩增效率从获得的30个多态性位点中筛选出22个微卫星位点用于亲权排除概率的计算，根据多态信息含量的大小由高到低依次增加位点数进行组合。结果表明，在两个亲本的基因型均未知的情况下，标记位点数为15个时，累积排除概率可达到99.99%，其中单个位点的第一非亲排除率（non-exclusion probability of the first parent，NE-1P）介于0.321~0.663之间。利用建立的亲子鉴定体系对16只具有系谱记录的小尾寒羊样本进行检测，结果共鉴定出4个具有高置信度的绵羊家系，鉴定结果与系谱记录完全一致。本试验为绵羊分子系谱的构建、亲子鉴定以及保障绵羊育种工作的正常开展奠定重要基础。

关键词: 绵羊; 分子遗传标记; 四碱基重复微卫星; 亲子鉴定; 基因分型

Abstract: The purpose of this study was to select the tetrameric microsatellite markers with high polymorphism,and to establish the paternity testing system of sheep.The Small-tail Han sheep was the main research object in this study,and fifty-three (ATAG)_n repeat microsatellite sites were selected according to the sheep reference genome sequence and a total of 30 microsatellite loci which had stable amplification and high polymorphism information were developed based on genotyping data in Small-tail Han sheep.The genotyping results of 30 loci showed that 253 alleles were obtained from the 30 markers,the average number of alleles was 8.433,the number of alleles was all greater than 5,the polymorphic information content (PIC) varied from 0.566 to 0.898,the calculated observed heterozygosity (Ho) ranged from 0.548 to 0.903 and the expected heterozygosity (He) ranged from 0.631 to 0.921 with a mean of 0.776.The Hardy-Weinberg equilibrium analysis revealed that all 30 sites were in genetic equilibrium.Then,22 of 30 microsatellite loci were selected according to the amplification efficiency of PCR for the calculation of parental exclusion probability,and the combinations were performed by increasing the number of bits from high to low according to the size of the PIC.The results showed that when the genotypes of the two parents were unknown,the cumulative probability of exclusion (CPE) could reach more than 99.99% when the number of marker sites was 15,and the NE-1P varied from 0.321 to 0.663.Further,16 Small-tail Han sheep with genealogical records were tested by using the established paternity test system,the results showed that 4 families with high confidence were identified,and the identification results were in complete agreement with pedigree records.This experiment laid an important foundation for the construction of sheep molecular genealogy,paternity test and the development of sheep breeding.

Key words: sheep; molecular genetic marker; tetrameric microsatellite markers; paternity analysis; genotyping

中图分类号:

S826.8⁺9

胡明月, 刘正喜, 赵中利, 曹阳, 马惠海, 闫守庆. 绵羊(ATAG)_n四碱基微卫星位点的筛选及其在亲子鉴定中的应用[J]. 中国畜牧兽医, 2021, 48(7): 2484-2494.

HU Mingyue, LIU Zhengxi, ZHAO Zhongli, CAO Yang, MA Huihai, YAN Shouqing. Isolation of (ATAG)_n Tetrameric Microsatellite Loci and Its Application on Paternity Analysis in Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(7): 2484-2494.

参考文献

[1] 殷彬,岳书俭,俞英,等.奶牛分子系谱构建或亲权鉴定的微卫星标记筛选[J].畜牧兽医学报,2017,48(4):595-604. YIN B,YUE S J,YU Y,et al.Selection of microsatellite markers used for pedigree tree construction and/or paternity testing in Chinese holstein cows[J].Acta Veterinaria et Zootechnica Sinica,2017,48(4):595-604.(in Chinese)
[2] 赵新,王永,兰青阔,等.基于复合EST-SSR标记的大白菜种子纯度鉴定及SNP位点获取[J].中国蔬菜,2013,14:31-38. ZHAO X,WANG Y,LAN Q K,et al.Purity identification and SNP site obtain of Chinese cabbage hybrids using multiplex EST-SSR marker[J].China Vegetables,2013,14:31-38.(in Chinese)
[3] 段燕梅.秦岭金丝猴(Rhinopithecus roxellana)基因组微卫星位点的筛选及亲子鉴定[D].西安:西北大学,2017. DUAN Y M.Characterization of highly polymorphic microsatallite markers and paternity analysis in the golden snub-nosed monkey (Rhinopithecus roxellana)[D].Xi'an:Northwest University,2017.(in Chinese)
[4] 王希彪,牟春堂,郝小燕,等.杜民杂交猪基因杂合度与繁殖性状关系研究[J].东北农业大学学报,2015,46(9):83-86. WANG X B,MU C T,HAO X Y,et al.Relationship between genetic heterozygosity and reproductive traits in Du Min hybridization pigs[J].Journal of Northeast Agricultural University,2015,46(9):83-86.(in Chinese)
[5] 梁家充,朱兰,孙利民,等.云南地方绵羊品种遗传多样性分子评价[J].四川农业大学学报,2018,36(6):822-830. LIANG J C,ZHU L,SUN L M,et al.Molecular evaluation of genetic diversity of native sheep breeds in Yunnan province[J].Journal of Sichuan Agricultural University,2018,36(6):822-830.(in Chinese)
[6] 李彬,任亭亭,邱梅玉,等.基于微卫星多重PCR技术的特克塞尔×哈萨克杂交羊亲子鉴定[J].中国畜牧兽医,2020,47(7):2171-2180. LI B,REN T T,QIU M Y,et al.Paternity identification of Texel×Kazakh sheep based on microsatellite multiple PCR technology[J].China Animal Husbandry & Veterinary Medicine,2020,47(7):2171-2180.(in Chinese)
[7] KAIRAT D,ZARINA O,AIZHAN M,et al.Genetic diversity of different breeds of Kazakh sheep using microsatellite analysis[J].Archives Animal Breeding,2019,62(1):305-312.
[8] 王吉振,王爱国,储明星,等.二核苷酸微卫星PCR扩增的影子带及其来源[J].畜牧兽医学报,2007,40(3):241-246. WANG J Z,WANG A G,CHU M X,et al.Shadow bands in PCR amplification of dinucleotide microsatellites and their origin[J].Acta Veterinaria et Zootechnica Sinica,2007,40(3):241-246.(in Chinese)
[9] 武辉,张思仲,肖翠英.三核苷酸重复序列PCR扩增中影子带的产生及其消除的方法[J].中华医学遗传学杂志,1998,1:3-5. WU H,ZHANG S Z,XIAO C Y.Shadow bands in PCR amplification of trinucleoyide repeats and their elimination[J].Chinese Journal of Medical Genetics,1998,1:3-5.(in Chinese)
[10] LEE M H,PARK S Y,KIM D J,et al.Genetic variation of three autosomal STR loci D21S1435,D21S1411,and D21S1412 in Korean population[J].Molecular Biology Reports,2010,37(1):99-104.
[11] LV D J.Detecting haplotypes of DXS7132 and DXS6804 loci by multiplex PCR[J].Journal of Genetics and Genomics,2003,30(1):10-14.
[12] SCHUELKE M.An economic method for the fluorescent labeling of PCR fragments[J].Nature Biotechnology,2000,18(2):233-234.
[13] WING K,CHUNG Y K,WONG D M.Power of exclusion revisited:Probability of excluding relatives of the true father from paternity[J].International Journal of Legal Medicine,2002,116(2):64-67.
[14] BOTSTEIN D,WHITE R L,SKOLNICK M,et al.Construction of a genetic linkage map in man using restriction fragment length polymorphisms[J].American Journal of Human Genetics,1980,32(3):314-331.
[15] KALINOWSKI S T,TAPER M L,MARSHALL T C.Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment[J].Molecular Ecology,2007,16(5):1099-1106.
[16] 任鹏.基于粪便DNA的小麂亲权鉴定、婚配制及种群数量研究[D].金华:浙江师范大学,2016. REN P.The parentage test,mating system and population quantity of Muntiacus reevesi based on fecal DNA[D].Jinhua:Zhejiang Normal University,2016.(in Chinese)
[17] 王悦.SNP用于新疆绵羊亲子鉴定的生物信息学方法研究[D].乌鲁木齐:新疆大学,2018. WANG Y.Bioinformatics methods for paternity test of sheep breeds in Xinjiang by SNP markers[D].Urumqi:Xinjiang University,2018.(in Chinese)
[18] 白云,蒙国华,刘瑞芳,等.番鸭微卫星标记的开发[J].中国家禽,2017,39(15):5-11. BAI Y,MENG G H,LIU R F,et al.Development of microsatellite markers for Muscovy ducks(Cairina moschata)[J].China Poultry,2017,39(15):5-11.(in Chinese)
[19] 张正义,邢秀梅,胡鹏飞,等.微卫星标记及其在动物亲缘关系鉴定中的应用[J].基因组学与应用生物学,2018,37(4):1406-1412. ZHANG Z Y,XING X M,HU P F,et al.Microsatellite markers and their application in animal genetic relationship identification[J].Genomics and Applied Biology,2018,37(4):1406-1412.(in Chinese)
[20] 高焕,于飞,栾生,等.中国明对虾基因组微卫星重复单元类型与其多态性关系[J].水生生物学报,2009,33(1):94-102. GAO H,YU F,LUAN S,et al.Relationships between polymorphism and types of SSR motif in the genome of Chinese shrimp Fenneropenaeus chinensis[J].Acta Hydrobiologica Sinica,2009,33(1):94-102.(in Chinese)
[21] VIGUERA E,CANCEILL D,EHRLICH S D.In vitro replication slippage by DNA polymerases from thermophilic organisms[J].Journal of Molecular Biology,2001,312(2):323-333.
[22] SHENG X,WANG Y,ZHANG J S,et al.Forensic investigation of 23 autosomal STRs and application in Han and Mongolia ethnic groups[J].Forensic Sciences Research,2018,3(2):138-144.
[23] EDWARDS A,HAMMOND H A,JIN L,et al.Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups[J].Genomics,1992,12(2):241-253.
[24] 熊飞,刘红艳,段辛斌,等.长薄鳅基因组四碱基重复微卫星的分离及序列特征分析[J].华中师范大学学报(自然科学版),2013,47(6):824-829. XIONG F,LIU H Y,DUAN X B,et al.Isolation and characterization of tetra-nucleotide repeat microsatellites from Leptobotia elongate[J].Journal of Central China Normal University(Natural Sciences),2013,47(6):824-829.(in Chinese)
[25] KANDPAL R P,KANDPAL G,WEISSMAN S M.Construction of libraries enriched for sequence repeats and jumping clones,and hybridization selection for region-specific markers[J].Proceedings of the National Academy of Sciences,1994,91(1):88-92.
[26] 熊良伟,王帅兵,封琦,等.基于高通量测序的中华鳑鲏基因组微卫星特征分析及标记开发[J].江苏农业科学,2018,46(18):164-168. XIONG L W,WANG S B,FENG Q,et al.Characteristic analysis of genomic microsatellites and development of SSR markers of Chinese bitterling (Rhodeus sinensis) using high-throughput sequencing[J].Jiangsu Agricultural Science,2018,46(18):164-168.(in Chinese)
[27] 杨坤,高源,邬明丽,等.伊犁马LTF基因多态性与产奶量相关性分析[J].家畜生态学报,2018,39(3):24-28. YANG K,GAO Y,WU M L,et al.Polymorphism of LTF gene and its correlation with lactation performance in Yili horse[J].Journal of Domestic Animal Ecology,2018,39(3):24-28.(in Chinese)
[28] PURVIS I W,FRANKLIN I R.Major genes and QTL influencing wool production and quality:A review[J].Genetics Selection Evolution,2005,37(Suppl 1):S97-107.
[29] 张瑞,郎侠,吴建平,等.岷县黑裘皮羊微卫星遗传多样性分析[J].中国畜牧兽医,2019,46(8):2360-2370. ZHANG R,LANG X,WU J P,et al.Genetic diversity analysis of microsatellite in Minxian Black fur sheep[J].China Animal Husbandry & Veterinary Medicine,2019,46(8):2360-2370.(in Chinese)
[30] 王可,刘昭华,曹洪防,等.山东省济宁青山羊群体遗传多样性分析[J].山东农业科学,2019,51(5):129-134. WANG K,LIU Z H,CAO H F,et al.Genetic diversity analysis of Jining Grey goat population in Shandong province[J].Shandong Agricultural Sciences,2019,51(5):129-134.(in Chinese)
[31] 王敏,张新浩,崔冉,等.利用微卫星标记鉴定德州驴亲子关系[J].中国畜牧兽医,2019,46(7):2003-2011. WANG M,ZHANG X H,CUI R,et al.Parentage testing of Dezhou donkey with microsatellite marker[J].China Animal Husbandry & Veterinary Medicine,2019,46(7):2003-2011.(in Chinese)
[32] 赵志达,王欣悦,石田培,等.基于微卫星标记鉴定湖羊家系[J].中国畜牧兽医,2020,47(11):3573-3583. ZHAO Z D,WANG X Y,SHI T P,et al.Identification of Hu sheep family based on microsatellite markers[J].China Animal Husbandry & Veterinary Medicine,2020,47(11):3573-3583.(in Chinese)