利用STR位点进行牛亲子鉴定的研究

为利用STR分型技术进行牛亲子鉴定,运用PCR技术用TGLA227、BM2113、TGLA53、ETH10、SPS115、TGLA126、TGLA122、INRA23、ETH3、ETH225、BM1824共11个STR(短串联重复序列)基因座对争议小牛、1号嫌疑母牛、2号嫌疑母牛的DNA进行扩增,扩增产物经遗传分析仪检测,检测结果用GeneMapper片段分析软件分析。鉴定结果可以排除1号母牛与争议小牛间具有亲子关系,不排除争议小牛与2号母牛间具有亲子关系。结果表明:STR基因分型技术能够准确、快速地进行牛亲子鉴定,该研究所用的STR位点具有较高的鉴别效率。     

牦牛10个STR基因座遗传多态性研究

利用PCR技术和全自动毛细血管电泳法技术检测牦牛BM1818、BM1824、CSSM66、TGLA53、ETH10、TGLA126、INRA037、ILSTS006、ILST0013、ETH225等10个STR基因座的多态性分布,并计算该10个基因座的基因频率(Pi)、杂合度(H)和多态信息含量(PIC)等值。结果显示:68只牦牛的10个微卫星座位共检测到102个等位基因,平均为10.2;牦牛中10个STR基因座的平均多态信息含量为0.685,10个基因座的平均多态信息含量大于0.5。其中10个基因座的平均观望杂合度和平均期望杂合度分别为0.7995、0.6154。属于高度杂合标记,遗传变异丰富。筛选出的10个STR基因座可用于牦牛遗传多样性、遗传结构分析及遗传图谱的构建等工作。     

微卫星技术在犬亲子鉴定中的应用一例

用PEZ1、FHC2054、FHC2010、PEZ5、PEZ20、PEZ12、PEZ3、PEZ6、PEZ8和FHC2079等10个STR基因座,对5头嫌疑公犬的血液样本DNA进行了多重PCR扩增,扩增产物经ABI3130遗传分析仪自动化检测.结果:在5头嫌疑公犬中找出了已知母本的一头仔犬的生物学父亲,其父权概率为99.9 999%.     

STR技术在种用东北细毛羊遗传管理中的应用

以10个STR基因座作为遗传标记系统，分析东北细毛羊种群的遗传多样性和亲缘关系。在遗传多样性分析中，群体的平均等位基因数为8．2，平均杂合度为0．794，平均多态信息含量为0．768，遗传多样水平较丰富。在亲缘关系鉴定和确认中，双亲鉴定时，10个微卫星基因座的累积非父排除概率为0．9999，平均父权相对机会(RCP)为99．999％；单亲鉴定时，累积非父排除概率为0．9962，平均RCP为99．993％。根据所选的STR基因座，可建立一套适用于种用东北细毛羊遗传资源分析和亲缘关系鉴定的方法体系，为我国东北细毛羊品种资源的保护和经济性状的筛选提供科学指导。同时，也可为解决东北细毛羊归属纠纷提供一个科学的鉴定方法。     

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。     

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。     

早胜牛遗传多态性与母系遗传背景分析

[目的]为评价和挖掘早胜牛遗传资源及种质资源,开展了早胜牛母系遗传背景及分子遗传特性研究。[方法]以早胜牛mtDNA D-Loop区为标记位点,对基因组DNA进行了PCR扩增和测序;以8个中国地方黄牛品种和5个引进品种的mtDNA D-Loop区核苷酸序列为对照,分析了核苷酸多态位点、核苷酸多样性、单倍型数等遗传多态性指标,构建了系统发育树。[结果]早胜牛及其杂交类群mtDNA D-Loop区富含A和T,检测到80个核苷酸变异位点,存在转换、颠换、转换和颠换共存3种突变类型,且以转换为主;界定了59个单倍型,其中8个共享单倍型,51个特有单倍型;我国地方黄牛分为两大支系,多数与非洲瘤牛、欧洲普通牛聚为一类,少数与印度瘤牛聚为一类;与其他地方黄牛品种相比,早胜牛及其杂交类群与秦川牛的亲缘关系最近。[结论]早胜牛及其杂交类群mtDNA D-Loop区核苷酸变异丰富,群体变异程度较高,与秦川牛的亲缘关系最近。     

湘东黑山羊BM1329微卫星标记的遗传多态性研究

利用经筛选的绵羊微卫星引物BM1329,采用聚丙烯酰胺凝胶垂直板电泳,对湘东黑山羊的BM1329微卫星基因座进行了多态性检测。结果表明:该基因座有8个等位基因,其片段大小在175bp~215bp之间,计算了该基因座各等位基因频率、基因型频率及其平均基因杂合度(0.8305)、多态信息含量(0.8090)和有效等位基因数(5.9001)。说明在湘东黑山羊中微卫星标记BM1329基因座具有丰富的遗传多态性。     

湘东黑山羊BM1329微卫星标记的遗传多态性研究

利用经筛选的绵羊微卫星引物BM1329,采用聚丙烯酰胺凝胶垂直板电泳,对湘东黑山羊的BM1329微卫星基因座进行了多态性检测.结果表明:该基因座有8个等位基因,其片段大小在175bp～215bp之间,计算了该基因座各等位基因频率、基因型频率及其平均基因杂合度(0.8305)、多态信息含量(0.8090)和有效等位基因数(5.9001).说明在湘东黑山羊中微卫星标记BM1329基因座具有丰富的遗传多态性.     

Resolution of parentage in dogs by examination of microsatellites after death of putative sire: case report.

A case of disputed paternity in dogs is reported. DNA examinations were carried out from hair samples of the individuals several months after the death of the putative sire. Ten short tandem repeat (STR) loci were analysed by fluorescence-labelled multiplex PCR using ABI PRISM 310 Genetic Analyser. Based on the results the candidate sire was included in the pedigree records as the biological sire. In spite of the genetic homogeneity of pedigree dogs due to inbreeding, canine microsatellites can provide an adequate basis for assigning paternity in pure breeds.     

Evaluation of recent changes in genetic variability in Japanese thoroughbred population based on a short tandem repeat parentage panel

The integrity of thoroughbreds is maintained under strict regulation involving DNA parentage testing, which is robust in a population with high genetic variability. The genetic variability of the thoroughbred population is possibly fluctuating because of selective breeding that has focused on adaptations for racing performance. To monitor genetic variability within the population and the effectiveness of short tandem repeat (STR) parentage testing, we investigated allele frequencies and the exclusion probability (PE) of 16–17 loci of a parentage panel in the Japanese thoroughbred population over 15 years. Expected heterozygosities (He) of 14 loci indicated a decreasing trend, and the average He of the population decreased significantly. Low genetic variability was possibly induced by a decrease in population size and a selective breeding bias. Four loci showed both a significant increase in allele frequency and a significant decrease in He; it is assumed that those loci were affected by positive selection for racing performance. There was a significant decrease in the PE because of the changes in genetic variability; however, it has remained over 0.99995. The current STR panel is still effective for parentage control, but it will be necessary to continuously monitor genetic variability, which has decreased over 15 years.     

First case of sterility associated with sex chromosomal abnormalities in a jenny

Chromosomal abnormalities are one of the main causes of genetic infertility in horses. Currently, their detection rate is rising due to the use of new diagnostic tools employing molecular markers linked to the sex chromosome pair. Despite genetic similarities, there are no previous reports of sterility associated with chromosomal abnormalities in the domestic donkey (Equus asinus). Hereby, we determined the presence of a chromosomal mosaicism in a female donkey with reproductive problems using molecular methodologies developed for horses. A two‐and‐a‐half‐year‐old jenny characterized by morphological abnormalities of the reproductive tract was cytogenetically analysed using conventional and fluorescent techniques and a group of microsatellite markers (short tandem repeat, STR). At the same time, five ultrasound measures of the reproductive tract were taken and compared with eight contemporary jennies of the same breed. After slaughter, morphological examinations showed that the case study had a blind vaginal vestibule defining an empty pouch that covered the entrance of the cervical os. Histopathological studies demonstrated that this abnormal structure was compatible with a remnant hymen. Molecular markers, STR and fluorescent in situ hybridization determinations revealed that the animal was a 62, XX/61,X mosaic and, therefore, the first case of chromosomal abnormalities in the sex pair reported in donkeys.     

8个微卫星位点在甘肃高山细毛羊肉毛兼用品系中的遗传多样性研究

利用8个微卫星位点对甘肃高山细毛羊肉毛兼用品系进行遗传检测,计算了等位基因频率、有效等位基因数、群体杂合度和多态信息含量。结果表明,位点BM3501的有效等位基因数、群体杂合度和多态信息含量都最高（Ne=7.30,H=0.866,PIC=0.848）;位点BM3413的有效等位基因数、群体杂合度和多态信息含量都最低（Ne=2.33,H=0.573,PIC=0.478）;8个微卫星位点在甘肃高山细毛羊肉毛兼用品系中多态性丰富,除位点BM3413表现为中度多态外,其余位点均表现为高度多态。因此,甘肃高山细毛羊肉毛兼用品系遗传杂合度较高,遗传多样性丰富,选择余地较大;8个微卫星位点可用于甘肃高山细毛羊肉毛兼用品系遗传多样性评估,并可用于对其羊毛性状和肉用性状的进一步研究。     

Molecular characterization of Mycobacterium avium subsp. paratuberculosis using multi-locus short sequence repeat (MLSSR) and mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) typing methods

The aim of this study was to characterize 38 bovine strains of Mycobacterium avium subsp. paratuberculosis isolated in Ireland using 11 multi locus short sequence repeat (MLSSR) loci and 8 mycobacterial interspersed repetitive units-variable number of tandem repeat (MIRU-VNTR) loci. The discriminatory power of these two typing methods alone and combined was evaluated, as was the epidemiology of the isolates and the genotypes obtained. Using the MIRU-VNTR typing method (8 loci analysed), only 3 subtypes were detected with a discrimination index (DI) of 0.54, but one MIRU-VNTR type has not been identified in other studies. In contrast the MLSSR method (using 11 loci) differentiated the 38 MAP isolates into 18 types with DI of 0.92. Among these 18 types 6 have not been recorded previously. The combination of the 2 methods (MIRU-VNTR and MLSSR) produced 22 distinct genotypes giving a maximal DI of 0.94. According to the allelic diversity, some markers are more polymorphic than others and must be applied in priority for the differentiation of MAP bovine isolates. This is the first report of genotyping data for MAP on the island of Ireland and will be very useful for analysing its epidemiology, transmission and virulence.     

Evaluation of variable number tandem repeat (VNTR) loci in molecular typing of Mycobacterium bovis isolates from Ireland

Various sets of short tandem repeats such as the exact tandem repeats (ETRs), mycobacterial interspersed repetitive units (MIRUs) and variable number tandem repeat (VNTR) loci, have recently been described as effective tools in strain typing M. tuberculosis complex isolates, representative of global diversity. This study extends our previous study, evaluating the discrimination of a further 17 MIRU_VNTR loci individually and comparing the resolution of published VNTR sets and spoligotyping using a panel of 47 local M. bovis field isolates, including known epidemiologically linked isolates and 9 M. tuberculosis complex reference isolates. Individual loci differed greatly in their discrimination. The discriminatory capacity of novel combinations of the most discriminating VNTR loci was also assessed. In the panel of 47 M. bovis isolates, 17 unique profiles were resolved using VNTR set 1, whilst the MIRUs and ETRs resolved the panel into 11 and 6 profiles, respectively. A novel combination of 10 highly discriminatory VNTRs was determined, which resolved 30 unique profiles. The configuration of a multi-locus VNTR-based assay and its ability to provide a flexible, convenient and high-resolution genotyping method is discussed. We suggest a panel of VNTR markers which may be widely suitable for molecular epidemiological studies of M. bovis. However, the number and combination of informative VNTR markers selected needs to be determined empirically with reference to locally prevalent strains and will depend on the epidemiological study requirements.     

Multiple autoimmune diseases syndrome in Italian Greyhounds: preliminary studies of genome-wide diversity and possible associations within the dog leukocyte antigen (DLA) complex

A disorder manifested by multiple autoimmune disorders, and resembling autoimmune polyendocrine syndrome type 2 (APS-2) in humans, may exist in Italian Greyhounds. The incidence of this disorder is increasing and its potential impact on the health of the breed is becoming of great concern. The aims of the present study were to document the existence of this syndrome, conduct a preliminary assessment of genetic diversity across the breed and within affected and unaffected dogs, determine whether the disorder associates with the dog leukocyte antigen (DLA) complex, and demonstrate similarities to APS-2 of humans. To these ends, information on disease, pedigrees, and blood or buccal swab samples were collected from affected and healthy Italian Greyhounds and extracted DNA analyzed. Analysis of Y chromosome markers and mitochondrial DNA sequences showed that Italian Greyhounds evolved from a single patriline and two major and four minor matrilines. A panel of 24 highly polymorphic simple tandem repeat (STR) markers across 20 autosomes demonstrated that affected and unaffected dogs were not distinguishable from the population as a whole by heterozygosity, F-statistics, and principal component analysis (PCA). However, analysis of allele frequencies at each STR loci identified regions of increased or decreased disease risk on four chromosomes. A similar genetic analysis using 109 single nucleotide polymorphisms (SNPs) across the DLA region showed differences between affected and unaffected dogs. PCA and zygosity mapping of DLA SNPs from unrelated dogs demonstrated two distinct subpopulations among the affected individuals. One population was very homozygous and the other closely resembled unaffected dogs in its heterozygosity, suggesting the evolution of a disease prone bloodline as a result of non-random selection. Exon 2 sequencing of the DLA class II genes demonstrated 5-8 alleles at each locus and 14 three loci haplotypes. Two specific haplotypes containing DRB1*00203 or DRB1*02901 were associated with increased disease risk in about one-third of affected dogs. However, high density SNP association mapping across the DLA region and CFA12 did not corroborate the association.     

辽宁绒山羊7个微卫星位点的遗传多样性分析

试验分析了7个微卫星基因座位OarAE101、OarJMP8、BM143、BM6506、BM1824、BM6438I、LSTS004在辽宁绒山羊4个家系中的遗传多态性。结果表明,7个微卫星标记中仅有4个微卫星位点(OarAE101、OarJMP8、BM143、BM6506)表现出了多态性。OarAE101位点多态信息含量最高,为0.727;BM143位点多态信息含量最低,为0.526。在所标记群体中平均有效等位基因数为3.249个;平均多态信息含量为0.635;遗传杂合度均值为0.638。说明辽宁绒山羊品种内的遗传变异处于一个较高的水平,遗传多样性丰富,选择潜力很大。     

Genetic diversity of Korean Bacillus anthracis isolates from soil evaluated with a single nucleotide repeat analysis

Bacillus (B.) anthracis, the etiological agent of anthrax, is one of the most genetically monomorphic bacteria species in the world. Due to the very limited genetic diversity of this species, classification of isolates of this bacterium requires methods with high discriminatory power. Single nucleotide repeat (SNR) analysis is a type of variable-number tandem repeat assay that evaluates regions with very high mutation rates. To subtype a collection of 21 isolates that were obtained during a B. anthracis outbreak in Korea, we analyzed four SNR marker loci using nucleotide sequencing analysis. These isolates were obtained from soil samples and the Korean Center for Disease Control and Prevention. The SNR analysis was able to detect 13 subgenotypes, which allowed a detailed evaluation of the Korean isolates. Our study demonstrated that the SNR analysis was able to discriminate between strains with the same multiple-locus variable-number tandem repeat analysis genotypes. In summary, we obtained SNR results for four SNR marker loci of newly acquired strains from Korea. Our findings will be helpful for creating marker systems and help identify markers that could be used for future forensic studies.     

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.