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

Paternity Identification of Texel×Kazakh Sheep Based on Microsatellite Multiple PCR Technology

本研究旨在建立一套适用于特克塞尔×哈萨克杂交羊亲子关系的鉴定体系。试验选取11个微卫星标记位点进行组合扩增,通过优化微卫星标记位点组合的引物浓度、退火温度及反应体系等条件,建立了4组多重PCR体系,对多重PCR扩增产物采用毛细管电泳进行基因型分型,经PROSize3.0软件读取基因型分型结果,Cervus 3.0软件分析群体的遗传多样性,鉴定特克塞尔×哈萨克级进F2代(特哈级进F2)和横交F2代(特哈横交F2)的亲子关系。结果表明,特哈级进F2和特哈横交F2的等位基因数为180和140、平均等位基因数为16.364和12.727、平均观测杂合度(Ho)为0.533和0.544、平均期望杂合度(He)为0.807和0.831、平均多态信息含量(PIC)为0.783和0.803。对特哈级进F2和特哈横交F2两个品种70只候选亲本和64只候选子代的11个微卫星标记位点进行亲子关系鉴定,结果表明:当双亲基因型未知时的累积排除概率(CE-1P)为0.9995和0.9997;当单亲基因型已知时的累积排除概率(CE-2P)均达到1.0000;双亲基因型已知的累计排除概率(CEPP)均达到1.0000。说明选择的11个微卫星标记位点具有高度的多态性和较高的排除概率,适用于遗传分析和个体的亲子鉴定。利用微卫星标记建立的特克塞尔×哈萨克杂交羊亲子鉴定体系为分析绵羊群体遗传多样性和辅助育种工作提供了理论依据。

The purpose of this study was to establish a set of identification system for parent-child relationship of Texel×Kazakh sheep.In the experiment,11 microsatellite marker sites were selected for combined amplification.By optimizing the primer concentration,annealing temperature,and reaction system of the combination of microsatellite marker sites,4 sets of multiplex PCR systems were established.Capillary electrophoresis was performed after multiplex PCR to perform genotyping to identify.The genotyping results were read by PROSize 3.0 software,and the gene-tic diversity of the population was analyzed by Cervus 3.0 software.The parent-child relationship between Texel×Kazakh progressive F2 generation(Texel×Kazakh progressive F2)and cross F2 generation(Texel×Kazakh cross F2).The results showed that the alleles of the Texel×Kazakh progressive F2 and cross F2 were 180 and 140,the average numbers of alleles were 16.364 and 12.727,the average observed heterozygosity were 0.533 and 0.544,the average expected heterozygosity were 0.807 and 0.831,and the average polymorphism information content were 0.783 and 0.803.Parent-child relationship identification was performed at 11 microsatellite marker sites of 70 candidate parents and 64 candidate progenies of the Texel×Kazakh progressive F2 and cross F2 two breeds.The results showed that:When the parental genotype was unknown,the combined exclusion probability(CE-1P)was 0.9995 and 0.9997;when the parental genotype was known,the combined exclusion probability(CE-2P)was all reached 1.0000;And the combined exclusion probability of both parents reached 1.0000.It indicates that the 11 selected microsatellite marker sites had high polymorphism and high probability of exclusion,which was suitable for genetic analysis and paternity identification of individuals.The parent-child identification system of Texel×Kazakh sheep established by microsatellite markers provided a theoretical basis for analyzing genetic diversity of sheep populations and assisting breeding work.