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

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。

Isolation of (ATAG)n Tetrameric Microsatellite Loci and Its Application on Paternity Analysis in Sheep

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.