中国荷斯坦种公牛BLAD遗传缺陷的分子检测及系谱分析

本试验运用限制性片段长度多态性聚合酶链式反应（RFLP-PCR）方法,检测我国荷斯坦种公牛白细胞粘附缺陷（bovine leukocyte adhesion deficiency,BLAD）基因的携带频率。共检测了来自全国14个公牛站的587头种公牛,发现BLAD携带者8头,携带率为1.36%。对现有公牛系谱信息分析显示,携带者公牛来自美国、加拿大和中国,其中6头携带者公牛可以追溯到共同祖先Osborndale Ivanhoe。此外,本研究还对我国荷斯坦牛遗传缺陷的控制和携带者公牛的利用提出建议。     

北京地区荷斯坦牛BLAD遗传缺陷基因的分子筛查

牛白细胞黏附缺陷症(BLAD)是由常染色体上CD18基因单碱基突变(A→G)引起的隐性遗传疾病,隐性基因纯合时导致白细胞表面的β2整合素表达明显减少或缺乏而引起临床发病,患病牛的主要特征是机体免疫力降低、易患病,从而影响生产性能的表现,可严重影响奶牛场的经济效益。本研究利用PCR-SSCP方法对北京地区246头公牛样本和409头母牛样本进行了检测分析,研究结果表明,在所检测的样本中,荷斯坦种公牛(含后备公牛)和母牛BLAD携带率分别为0.81%和3.91%,BLAD基因频率分别为0.21%和1.95%,并通过系谱追踪发现,BLAD遗传缺陷可追溯到美国一头非常优秀的公牛"Osborndale Ivanhoe"(USA.1189870,BL),其后代在我国有一定的影响。通过剔除BLAD携带者公牛可以有效控制该遗传缺陷的传播,但是,我国部分BLAD携带者公牛冻精依然在商业化使用,所以,有效监控BLAD携带者在奶牛群中的状况对BLAD剔除计划是有益的。     

牛白细胞黏附缺陷病液相基因芯片检测方法的建立

从福建某奶牛场200头进口荷斯坦牛采血提取牛血液DNA，根据已知牛染色体上CD18编码基因序列383位基由A变为G而引起牛白细胞黏附缺陷症（BLAD）设计特异性野生型和突变型引物，建立液相基因芯片检测方法用于牛白细胞黏附缺陷症（BLAD）的检测，结果检出2头母牛为BLAD携带者，检出率为0．67％，没有发现患病牛。结果证明建立的液相基因芯片检测方法是一种敏感性和特异性很高的筛选奶牛BLAD有害基因的新方法     

北京地区荷斯坦牛白细胞黏附缺陷症的检测分析

牛白细胞黏附缺陷症(BLAD)是由常染色体上CD18基因单碱基突变(A→G)引起的隐性遗传疾病,患病牛只机体免疫力降低、易患病,影响其生产性能的表现。本研究采用限制性片段长度多态性聚合酶链反应方法(PCR-RFLP),检测了北京502头荷斯坦母牛CD18编码基因第二外显子的多态性。结果表明,3个奶牛场中共检测到2种基因型——AA和AG,未检测到GG基因型。检测群体BLAD基因携带率为3.1%,说明北京地区规模化奶牛场使用的公牛冻精中BLAD基因没有得到彻底净化,导致母牛群体中BLAD基因携带率在3.0%以上。在奶牛育种和生产中,应有计划的淘汰携带BLAD基因的个体,净化京郊荷斯坦奶牛群体。     

牛白细胞黏附缺陷病等位基因频率分析

为了解荷斯坦奶牛群中成母牛白细胞黏附缺陷病情况,抽取1 000头牛血样,采用RT-PCR方法对血样进行分析,并用TaqⅠ内切酶对RT-PCR产物进行消化,再用1.2%琼脂糖凝胶分析Taq Ⅰ限制性位点酶切片段.结果表明:1 000头荷斯坦奶牛中有19头BLAD基因携带者,1头BLAD基因纯合个体;BLAD基因携带者和纯合个体所占的比率分别为1.9%和0.1%;无BLAD和携带BLAD的基因频率分别为0.989 5和0.010 5.     

中国荷斯坦公牛CN和DUMPS遗传缺陷检测及系谱分析

旨在研究中国荷斯坦牛中瓜氨酸血症(Citrullinemia,CN)和尿苷酸核酶缺乏症(Deficiency of uridinemonophosphate synthase,DUMPS)2种遗传缺陷的携带者比率及系谱来源,并构建更简便的检测方法。本研究通过PCR-RFLP方法对参加我国联合青年公牛后裔测定和良种补贴项目的591头荷斯坦公牛进行了大规模CN和DUMPS的遗传缺陷检测,并构建了奶牛CN隐性有害基因的AS-PCR检测技术。结果,共发现2头CN和1头DUMPS隐性有害基因携带者公牛,携带者比例分别为0.34%和0.17%。经过系谱追溯,2头CN携带者公牛均为澳大利亚公牛Linmack Kriss King-CN后代,DUMPS携带者公牛为美国公牛Skokie sensation Ned后代。基于此,我国有必要尽快建立荷斯坦牛隐性遗传缺陷监控体系并进行系谱标注,通过青年公牛预选和选种选配,避免携带者公牛进入后裔测定和良种补贴项目,以逐步降低我国奶牛群体中隐性有害等位基因频率。     

天津地区荷斯坦公牛CVM、DUMPS和CN遗传缺陷检测

本文旨在研究天津地区中国荷斯坦公牛脊椎畸形综合征（Complex vertebral malformation,CVM）、尿苷酸合酶缺乏症（Deftciencv of uridine monophospham synchase,DUMPS）和瓜氨酸血症（Citrullinemia,CN）3种遗传缺陷的携带者比率及系谱来源。通过PIRA—PCR和PCR—RFLP方法分别对天津奶牛发展中心参加全国青年公牛联合后裔测定和国家良种补贴项目的110头荷斯坦公牛进行了CVM、DUMPS和CN三种遗传缺陷检测。共发现6头CVM隐性有害基因携带公牛,携带者比例为5．45％,隐性有害等位基因频率为2．72％。经过系谱分析,其中4头CVM携带者均为美国公牛Carlin—MIvanhoeBell的后代,另外2头因系谱不完整而无法查询。未检测到DuMPs和CN隐性有害基因携带者。基于此,我国有必要尽快建立荷斯坦牛隐性遗传缺陷监控体系并进行系谱标注,避免携带公牛进入后裔测定和良种补贴项目,以逐步降低我国奶牛群体中遗传缺陷隐性等位基因频率。     

中国荷斯坦牛凝血因子XI缺陷症遗传分析

凝血因子XI缺陷症（FactorXIdenciency）是荷斯坦牛的一种常染色体单基因控制的隐性遗传缺陷。该病的遗传基础是由位于牛第27号染色体的凝血因子XI基因外显子12上发生的一段76bp序列插入。本研究采用PCR方法对我国13个主要公牛站的571头荷斯坦公牛的凝血因子XI基因进行了全面检测,未发现隐性有害基因携带者和纯合个体。     

奶牛隐陛遗传缺陷脊椎畸形综合征的SSCP分析

丹麦科学家1999年首先发现了荷斯坦牛中存在的、造成大量流产和畸形犊牛的脊椎畸形综合征（CVM）遗传缺陷。全世界奶牛业已经验证并认同丹麦科学家的研究结果，世界各国纷纷采取措施对付这种有史以来因遗传原因给荷斯坦牛造成巨大损失的疾病。我国奶牛群中也同样存在着隐性遗传缺陷CVM问题，可能已经给奶牛场造成巨大的损失。本文介绍了奶牛CVM的概况和CVM隐性遗传缺陷基因的分子检测方法，分析了其遗传机理和传递规律．以及国外对CVM的发现、检测和处理办法，介绍了控制CVM的关键点。北京奶牛中心在国内率先对本单位的150头荷斯坦种公牛和140头荷斯坦母牛进行DNA测定，对种公牛和母牛的DNA样品进行了SSCP分析，结果表明，在所检测的150头种公牛中，有16头是CVM携带者，携带率为10．7％；140头母牛中，17头是携带者，携带率为12％。通过基因检测技术对种公牛进行选择．向养牛户提供CVM非携带者的公牛精液，避免CVM隐性遗传缺陷基因造成的经济损失．从而提高奶牛场的经济效益。     

奶牛隐性遗传缺陷脊椎畸形综合征的SSCP分析

丹麦科学家1999年首先发现了荷斯坦牛中存在的、造成大量流产和畸形犊牛的脊椎畸形综合征(CVM)遗传缺陷.全世界奶牛业已经验证并认同丹麦科学家的研究结果,世界各国纷纷采取措施对付这种有史以来因遗传原因给荷斯坦牛造成巨大损失的疾病.我国奶牛群中也同样存在着隐性遗传缺陷CVM问题,可能已经给奶牛场造成巨大的损失.本文介绍了奶牛CVM的概况和CVM隐性遗传缺陷基因的分子检测方法,分析了其遗传机理和传递规律.以及国外对CVM的发现、检测和处理办法.介绍了控制CVM的关键点.北京奶牛中心在国内率先对本单位的150头荷斯坦种公牛和140头荷斯坦母牛进行DNA测定,对种公牛和母牛的DNA样品进行了SSCP分析,结果表明,在所检测的150头种公牛中,有16头是CVM携带者,携带率为10.7%:140头母牛中,17头是携带者,携带率为12%.通过基因检测技术对种公牛进行选择.向养牛户提供CVM非携带者的公牛精液,避免CVM隐性遗传缺陷基因造成的经济损失,从而提高奶牛场的经济效益.     

Comparison of milk production of the progeny of BLAD-carrier and healthy Holstein bulls in Hungary.

'Bovine Leukocyte Adhesion Deficiency' (BLAD) is a recessive monofactorial, lethal inheritable defect occurring in Holstein-Friesian cattle and often passed on by well-known top bulls. The aim of this study was to find a relationship between the BLAD genotype of bulls, their genetic evaluation for milk and their daughters' milk production. BLAD-carrier and healthy bulls were compared on the basis of their breeding value published in November 1997. The first 100 bulls ranked according to the Total Production Index (TPI) were used, including nine BLAD carriers with 2,835 daughters and 77 healthy sires with 21,950 female progenies. For 14 bulls the BLAD genotype was not indicated. The healthy animals significantly outperformed the BLAD carriers, which result contradicts our earlier findings (Dohy et al., 1996; Jánosa and Dohy, 1997). In a BLAD elimination programme, the identification of BLAD carriers and properly planned mating are of great importance in order to avoid 'inter se' mating of BLAD-carrier top animals which can be of significant influence in Holstein breeding.     

Bovine leukocyte adhesion deficiency (BLAD): a review

Bovine leukocyte adhesion deficiency (BLAD) in Holstein cattle is an autosomal recessive congenital disease characterized by recurrent bacterial infections, delayed wound healing and stunted growth, and is also associated with persistent marked neutrophilia. The molecular basis of BLAD is a single point mutation (adenine to guanine) at position 383 of the CD18 gene, which caused an aspartic acid to glycine substitution at amino acid 128 (D128G) in the adhesion molecule CD18. Neutrophils from BLAD cattle have impaired expression of the beta2 integrin (CD11a,b,c/CD18) of the leukocyte adhesion molecule. Abnormalities in a wide spectrum of adherence dependent functions of leukocytes have been fully characterized. Cattle affected with BLAD have severe ulcers on oral mucous membranes, severe periodontitis, loss of teeth, chronic pneumonia and recurrent or chronic diarrhea. Affected cattle die at an early age due to the infectious complications. Holstein bulls, including carrier sires that had a mutant BLAD gene in heterozygote were controlled from dairy cattle for a decade. The control of BLAD in Holstein cattle by publishing the genotypes and avoiding the mating between BLAD carriers was found to be successful. This paper provides an overview of the genetic disease BLAD with reference to the disease in Holstein cattle.     

A novel method for rapid and reliable detection of complex vertebral malformation and bovine leukocyte adhesion deficiency in Holstein cattle

Background: Complex vertebral malformation (CVM) and bovine leukocyte adhesion deficiency (BLAD) are two autosomal recessive lethal genetic defects frequently occurring in Holstein cattle, identifiable by single nucleotide polymorphisms. The objective of this study is to develop a rapid and reliable genotyping assay to screen the active Holstein sires and determine the carrier frequency of CVM and BLAD in Chinese dairy cattle population. Results: We developed real-time PCR-based assays for discrimination of wild-type and defective alleles, so that carriers can be detected. Only one step was required after the DNA extraction from the sample and time consumption was about 2 hours. A total of 587 Chinese Holstein bulls were assayed, and fifty-six CVM-carriers and eight BLAD-carriers were identified, corresponding to heterozygote carrier frequencies of 9.54% and 1.36%, respectively. The pedigree analysis showed that most of the carriers could be traced back to the common ancestry, Osborndale Ivanhoe for BLAD and Pennstate Ivanhoe Star for CVM. Conclusions: These results demonstrate that real-time PCR is a simple, rapid and reliable assay for BLAD and CVM defective allele detection. The high frequency of the CVM allele suggests that implementing a routine testing system is necessary to gradually eradicate the deleterious gene from the Chinese Holstein population.     

A novel method for rapid and reliable detection of complex vertebral malformation and bovine leukocyte adhesion deficiency in Holstein cattle

Background

Complex vertebral malformation (CVM) and bovine leukocyte adhesion deficiency (BLAD) are two autosomal recessive lethal genetic defects frequently occurring in Holstein cattle, identifiable by single nucleotide polymorphisms. The objective of this study is to develop a rapid and reliable genotyping assay to screen the active Holstein sires and determine the carrier frequency of CVM and BLAD in Chinese dairy cattle population.

Results

We developed real-time PCR-based assays for discrimination of wild-type and defective alleles, so that carriers can be detected. Only one step was required after the DNA extraction from the sample and time consumption was about 2 hours. A total of 587 Chinese Holstein bulls were assayed, and fifty-six CVM-carriers and eight BLAD-carriers were identified, corresponding to heterozygote carrier frequencies of 9.54% and 1.36%, respectively. The pedigree analysis showed that most of the carriers could be traced back to the common ancestry, Osborndale Ivanhoe for BLAD and Pennstate Ivanhoe Star for CVM.

Conclusions

These results demonstrate that real-time PCR is a simple, rapid and reliable assay for BLAD and CVM defective allele detection. The high frequency of the CVM allele suggests that implementing a routine testing system is necessary to gradually eradicate the deleterious gene from the Chinese Holstein population.     

Detection of bovine leukocyte adhesion deficiency (BLAD) in Turkish native and Holstein cattle

The purpose of this work was to study whether the bovine leukocyte adhesion deficiency (BLAD) allele is present in native cattle breeds and the Holstein breed in Turkey. Blood samples were obtained from 120 Holstein, 20 Brown Swiss, 20 Anatolian Black, 20 Turkish Grey, 20 South Anatolian Red and 20 East Anatolian Red cattle. The isolated DNA materials were multiplied in PCR using the primer developed by Kriegesmann et al. (1997). In order to determine the area of mutation in PCR products, the PCR products were digested with TaqI endonuclease enzyme. The resulting fragments were analysed on 2% agarose gel for the absence of a TaqI restriction site. It was found that two of the Holstein cattle (a bull and a cow) were heterozygote BLAD carriers. There was no homozygote BLAD animal. The BLAD allele was not found in the other breeds used in the study. The mutant BLAD allele frequency in the 120 Holstein cattle calculations was 0.0084.     

Screening for bovine leukocyte adhesion deficiency,deficiency of uridine monophosphate synthase,complex vertebral malformation,bovine citrullinaemia,and factor XI deficiency in Holstein cows reared in Turkey

Background

Bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine monophosphate synthase (DUMPS), complex vertebral malformation (CVM), bovine citrullinaemia (BC) and factor XI deficiency (FXID) are autosomal recessive hereditary disorders, which have had significant economic impact on dairy cattle breeding worldwide. In this study, 350 Holstein cows reared in Turkey were screened for BLAD, DUMPS, CVM, BC and FXID genotypes to obtain an indication on the importance of these defects in Turkish Holsteins.

Methods

Genomic DNA was obtained from blood and the amplicons of BLAD, DUMPS, CVM, BC and FXID were obtained by using PCR. PCR products were digested with TaqI, AvaI and AvaII restriction enzymes for BLAD, DUMPS, and BC, respectively. These digested products and PCR product of FXID were analyzed by agarose gel electrophoresis stained with ethidium bromide. CVM genotypes were detected by DNA sequencing. Additionally, all genotypes were confirmed by DNA sequencing to determine whether there was a mutant allele or not.

Results

Fourteen BLAD, twelve CVM and four FXID carriers were found among the 350 Holstein cows examined, while carriers of DUMPS and BC were not detected. The mutant allele frequencies were calculated as 0.02, 0.017, and 0.006 for BLAD, CVM and FXID, respectively with corresponding carrier prevalence of 4.0% (BLAD), 3.4% (CVM) and 1.2% (FXID).

Conclusion

This study demonstrates that carriers of BLAD, CVM and FXID are present in the Turkish Holstein population, although at a low frequency. The actual number of clinical cases is unknown, but sporadic cases may appear. As artificial insemination is widely used in dairy cattle breeding, carriers of BLAD, CVM and FXID are likely present within the population of breeding sires. It is recommended to screen breeding sires for these defective genes in order to avoid an unwanted spread within the population.     

Identification of complex vertebral malformation carriers in Chinese Holstein.

Complex vertebral malformation (CVM) is a monogenic autosomal recessive hereditary defect of Holstein dairy cattle. It is caused by a point mutation from G to T at the nucleotide position 559 in bovine solute carrier family 35, member 3 gene (SLC35A3), which changes the amino acid sequence of uridine 5'-diphosphate-N-acetylglucosamine transporter protein from a valine to a phenylalanine in position 180. The elite U.S. Holstein sire Penstate Ivanhoe Star was identified as the common ancestor of the current CVM carriers. Because his offspring, mainly those of Carlin-M Ivanhoe Bell, were used in many countries, CVM has potentially spread into China. In the present study, using the polymerase chain reaction-single-stranded conformational polymorphism (PCR-SSCP) technique, 10 CVM carriers were found among 68 at-risk Chinese Holstein bulls, and 282 carriers were found among 602 at-risk cows. The results of this study indicate that the CVM gene exists in the Chinese Holstein population.