微卫星DNA标记在蓝狐单亲亲权鉴定中的应用

主要探讨微卫星DNA标记在蓝狐(Alopex lagopus)单亲亲权鉴定中的可行性,为蓝狐育种的系谱精确鉴定提供依据。选择15个多态信息含量较高的微卫星DNA位点,以6窝蓝狐(母本6只,子代61只)为试验材料,计算15个基因座位的等位基因频率、杂合度(H)、多态信息含量(PIC)、非父排除概率(EP)、累积非亲排除概率(CCE)、亲权指数(PI)和亲权相对机会(RCP)。结果表明:15个微卫星位点的累积非亲排除概率值(CCE)为0.999996。单亲鉴定的PI值为850.5213~966160,RCP为99.8826%~99.9999%。说明所选的15个微卫星位点可用于蓝狐单亲亲权鉴定,其判定成功率和准确率较高,结果可靠。     

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

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。     

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

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。     

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。     

中国四个海拔牦牛群体血红蛋白β链微卫星多态性分析

分析了四个海拔高度牦牛（4500m，4000m，3500m，1700m）血红蛋白β链微卫星座位的多态性，结果发现中国牦牛群体的平均杂合度和多态信息含量分别为0．8574，0．8426，等位基因数为10，其中7个是牦牛所特有的，另外3个是牦牛和黄牛所共有的。四个海拔高度牦牛的等位基因分布差异极显著（P〈0．01）。等位基因分布与海拔相关，相关系数为0．397。基因型121bp／103bp是海拔4000m处牦牛所特有的。这说明高度多态的血红蛋白β链微卫星座位在牦牛适应高原低氧中具有可能的自然选择价值及可能的调控作用。     

陇东绒山羊微卫星DNA遗传多样性的研究

利用9个微卫星座位,8％非变性聚丙烯酰胺凝胶电泳PCR扩增产物,对120只陇东绒山羊基因组DNA的遗传多样性进行了检测,对其群体遗传学特性进行分析。结果表明：陇东绒山羊在9个微卫星座位中共检测到90个等位基因,其中微卫星座位LSCV24等位基因数最多,为14个;BMS1248多态信息含量最高,PIC为0．88;DB6座位多态信息含量最低,PIC为0．79。在所标记群体中平均有效等位基因数为7．69±1．65个,平均多态信息含量为0．84±0．03;遗传杂合度均值为0．86±0．03,均高于国内外部分研究结果。说明陇东绒山羊品种遗传多样性丰富,群体遗传变异程度高,育种潜力大。     

长江三角洲白山羊群体遗传结构研究

应用结构基因座和微卫星DNA两种遗传标记探讨长江三角洲白山羊群体遗传结构。结果表明,检测的9个结构基因座位上7个存在多态性,座位的基因平均杂合度、多态信息含量及有效等位基因数分别为0.1767、0.1457和1.2837;7个微卫星位点上共检测到110个等位基因,位点的基因平均杂合度、多态信息含量及有效等位基因数分别为0.8867、0.8774和11.2907。群体内的遗传变异程度相对较高,反映出丰富的遗传多样性,而且微卫星DNA标记揭示的遗传变异高于结构基因座。     

长江三角洲白山羊群体遗传结构研究

应用结构基因座和微卫星DNA两种遗传标记探讨长江三角洲白山羊群体遗传结构。结果表明,检测的9个结构基因座位上7个存在多态性,座位的基因平均杂合度、多态信息含量及有效等位基因数分别为0．1767、0．1457和1．2837;7个微卫星位点上共检测到110个等位基因,位点的基因平均杂合度、多态信息含量及有效等位基因数分别为0．8867、0．8774和11．2907。群体内的遗传变异程度相对较高,反映出丰富的遗传多样性,而且微卫星DNA标记揭示的遗传变异高于结构基因座。     

利用微卫星标记鉴定德州驴亲子关系

试验旨在建立一套适用于德州驴亲子关系的鉴定体系。选取13个微卫星基因座作为标记,采集了53头德州驴血液样本,其中子代驴驹16头,候选父本13头,候选母本24头,用酚-仿法抽提血液基因组进行PCR扩增和基因扫描,并利用Peak Scanner Software v1.0软件读取基因型分型结果。对微卫星基因座的遗传多样性进行分析,利用似然法（Cervus 3.0软件）和排除法对个体间的亲子关系进行了鉴定。结果显示,13个微卫星基因座的平均等位基因数、平均观测杂合度（Ho）、平均期望杂合度（He）和平均多态信息含量（PIC）分别为6.846、0.689、0.671和0.625。期望杂合度与观测杂合度之差在0.002~0.088之间,差值较小。13个微卫星基因座的累计排除概率（EP）达到0.990以上。微卫星基因座具有高度多态性和较高的排除概率,适用于遗传分析和个体鉴定。利用Cervus 3.0软件基于似然法分析得到了16头子代驴驹的最似亲本,结合排除法对这16头驴驹及其最似亲本进行基因型比对,最终在53头德州驴中确定了11个亲子对。本试验建立了以13个微卫星位点作为核心标记,将似然法和排除法相结合作为主要分析方法的德州驴亲子关系鉴定体系,为育种工作提供参考资料。     

利用微卫星标记鉴定德州驴亲子关系

试验旨在建立一套适用于德州驴亲子关系的鉴定体系。选取13个微卫星基因座作为标记,采集了53头德州驴血液样本,其中子代驴驹16头,候选父本13头,候选母本24头,用酚-仿法抽提血液基因组进行PCR扩增和基因扫描,并利用Peak Scanner Software v1.0软件读取基因型分型结果。对微卫星基因座的遗传多样性进行分析,利用似然法(Cervus 3.0软件)和排除法对个体间的亲子关系进行了鉴定。结果显示,13个微卫星基因座的平均等位基因数、平均观测杂合度(Ho)、平均期望杂合度(He)和平均多态信息含量(PIC)分别为6.846、0.689、0.671和0.625。期望杂合度与观测杂合度之差在0.002～0.088之间,差值较小。13个微卫星基因座的累计排除概率(EP)达到0.990以上。微卫星基因座具有高度多态性和较高的排除概率,适用于遗传分析和个体鉴定。利用Cervus 3.0软件基于似然法分析得到了16头子代驴驹的最似亲本,结合排除法对这16头驴驹及其最似亲本进行基因型比对,最终在53头德州驴中确定了11个亲子对。本试验建立了以13个微卫星位点作为核心标记,将似然法和排除法相结合作为主要分析方法的德州驴亲子关系鉴定体系,为育种工作提供参考资料。     

Identification of Hu Sheep Family Based on Microsatellite Markers

The purpose of this study was to explore identification and division methods of Hu sheep family based on simple sequence repeats(SSR) polymorphism.Nine SSR loci of BM3051,HH64,TGLA137,MAF33,FCB48,VH72,INRA023,ETH152 and MCM527 were selected,which were distributed in several autosomes,with high polymorphism and high heterozygosity.DNA was extracted from the blood samples of 30 Hu breeding rams by blood DNA extraction kit.SSR primers were synthesized and labeled with the fluorescent group.The products were amplified by touch-down PCR.After genotyping,CERVUS3.0.7 was used for genotyping analysis.POPGENE1.32 was used to construct a dendrogram based on Nei's genetic distance to divide Hu sheep families.The results showed that the cumulative exclusion probability of single parent was 99.2094% when the genotypes of both parents were unknown,99.9688% when a single parent genotypes were known,99.9999% when both parents' genotypes were unknown.Exclusion probability could meet the requirements of paternity identification and pedigree analysis in genetic breeding.UPGMA clustering map was constructed according to Nei's genetic distance,and 30 breeding rams were divided into six families,which provided a reference for sheep breeding.The method could run the family identification and division of Hu sheep,which had application value for the breeding of Hu sheep.     

基于微卫星标记鉴定湖羊家系

试验旨在基于微卫星（simple sequence repeats，SSR）多态性探索一种鉴定与划分湖羊家系的方法，通过查阅文献筛选出9个分布于多个常染色体、具有高多态性信息、高杂合度的湖羊微卫星位点BM3051、HH64、TGLA137、MAF33、FCB48、VH72、INRA023、ETH152和MCM527。采集30只湖羊种公羊血液，使用试剂盒提取血液基因组DNA，合成筛选出的9个微卫星引物并使用荧光基团标记，利用降落式PCR进行扩增，扩增产物在基因分型后使用CERVUS3.0.7进行基因型分析，使用POPGENE1.32基于Nei氏遗传距离构建树状聚类图，根据图示的亲缘关系远近划分湖羊家系。结果显示，当双亲基因型未知时，9个微卫星位点的单亲累积排除概率为99.2094%；当单亲基因型已知时，9个微卫星位点的累积排除概率为99.9688%；当双亲基因型未知时9个微卫星位点的双亲累积排除概率为99.9999%，排除概率能够满足遗传育种中亲子鉴定和系谱分析的要求。根据Nei氏遗传距离构建UPGMA聚类图，将30只种公羊划分为6个家系，为育种工作提供了一定参考。本方法能够进行湖羊家系的鉴定与划分，对湖羊育种工作具有一定的应用价值。     

An Example of Yak Paternity Assignment by Microsatellite Genotyping

12 cattle microsatellite DNA markers were used for establishing the paternity of 4 Datong Yak calves.The results indicated that paternity can be assigned by use of exclusion method directely when the mother is clearly known and data of all likely sires were obtained. The more the alleles of a locus possessed, the more effectively the paternity can be assigned. Not only the microsatellite DNA marker is a promising tool in establishment of yak detail pedigrees, but also can provide us information of reproductive efficiency of the bull(s) based on the numbers of calves it produced in the herd, which will be useful in speeding up the processes of yak genetics improvement.     

Evaluation of parentage testing in the Turkish Holstein population based on 12 microsatellite loci

In the present study, 12 microsatellite loci (ETH10, ETH225, ETH03, TGLA122, TGLA227, BM1824, BM2113, INRA23, SPS115, TGLA126, RM006 and BM1818) were evaluated for their possible use to confirm selected pedigree relationships between 7 bulls, their 21 male offspring, and their 64 second-generation female offspring within the progeny test started in Turkey. The nine loci (BM1824, INRA23, BM2113, SPS115, ETH10, TGLA122, ETH225, TGLA126 and TGLA227) recommended by ISAG displayed high values for the measures of informativeness (allele numbers, heterozygosity, polymorphic information content, frequency of the most common allele, and power of discrimination). When both parents are known calculated combined probability of exclusion was at least 0.999. Range of probability of paternity (POP) values were 0.814–0.9999. Except 3 cases (4.7%), the alleged paternity relationships were confirmed. To have a higher confidence in POP values new loci must be integrated into the set of 9 loci used.     

Analysis of Two Chinese Yak(Bos grunniens) Population Using Bovine Microsatellite Primers

Two Chinese domestic yak populations representing the Plateau type and the Huanhu Alpine type were analysed with 12 bovine microsatellite primers. All primer pairs functioned in the yak genome and polymorphism was found at all loci. The allele size ranges and frequencies of the two yak populations were similar and there was considerable overlap with the allele size ranges observed in cattle. Data for European cattle breeds was obtained from the Cattle Diversity Database(CaDBase)to interpret the heterozygosity and genetic distance estimates in yak populations. Heterozygosity estimated for the two yak populations was comparable to that of European cattle while Nei's Genetic Distance DA between the two yak populations was less than distances between the most closely related German cattle breeds. Bovine microsatellite primers proved to be a valuable tool for characterization of yak populations.     

Characterization of Swiss new world camelid breeds using microsatellite markers

Molecular markers allow a characterization of animal populations on DNA level. They help to estimate the genetic variability, they are useful in paternity control, to diagnose hereditary diseases, in epidemiology and last but not least support traditional tools to take breeding decisions. Seventy-six new-world camelids in Switzerland were genetically typed for six microsatellite markers and their parentage controlled. Calculation of allele frequencies, heterozygosity, polymorphism information content, deviation from Hardy-Weinberg equilibrium and exclusion probability of markers showed a high genetic variability within the chosen populations. This will allow selection for certain traits. The estimated genetic distance between different camelid species and breeds was in accordance with their expected phylogenetic origins.     

利用微卫星和SNP标记信息进行奶牛亲子鉴定的模拟研究

本研究旨在比较微卫星标记和单核苷酸多态(SNP)标记进行奶牛亲子鉴定的效率。研究中随机模拟了10 000对含微卫星或SNP标记信息的非亲子关系的个体,用排除法进行亲子鉴定,该过程重复100次。结果表明,标记的多态性对于排除概率的影响最大。达到同样的排除概率,所需低多态标记数量远高于高多态标记。当真实母亲基因型已知时,个体父亲的推断效率较高。达到同样的排除概率,无论标记多态性高或低,3种鉴定类型所需SNP标记数量均大于微卫星标记。当SNP和微卫星标记的平均杂合度为0.484和0.875时,需要的SNP标记数是微卫星标记的5~6倍;当SNP和微卫星标记的平均杂合度为0.363和0.566时,需要的SNP标记数约是微卫星标记的2倍。在2种标记中,达到同样排除概率(大于0.99),采用至少2个矛盾推断原则推断所需标记数为单个矛盾推断原则的1.45倍。结果表明,由于SNP标记具有误判率低、重复性高和易于高通量自动化检测等特点,随着检测成本降低,SNP标记将来完全有可能取代微卫星标记用于奶牛群体的亲子鉴定。