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。     

北京地区荷斯坦牛脊椎畸形综合征的调查研究

脊椎畸形综合征（Complex Vertebral Malformation,CVM）是由常染色体上SLC35A3基因单碱基突变（G→T）引起的隐性遗传疾病,该基因隐性纯合（CV/CV）时奶牛致死,但CVM携带者表现正常,所以CVM携带者公牛可以通过人工授精技术传播CVM缺陷基因。本研究利用PCR-SSCP方法对北京地区242头公牛样品和403头母牛样品进行了检测分析,研究结果表明,在所检测的样本中,荷斯坦种公牛和母牛CVM携带率分别为8.82%和5.71%,CVM基因频率分别为4.41%和2.85%。通过系谱追踪发现,CVM遗传缺陷的共同祖先是美国名牛＂Penstate Ivanhoe Star＂（USA.1441440,CV）。通过剔除CVM携带者公牛可以有效地控制CVM遗传缺陷的传播,但是,我国许多CVM携带者公牛冻精依然在商业化使用,所以有效地监控CVM携带者在奶牛群中的状况对CVM防控计划是有益的。     

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.     

北京地区荷斯坦牛短脊椎综合征的分子筛查

牛短脊椎综合征(Brachyspina syndrome,BS)是在荷斯坦牛群中发现的一种隐性遗传缺陷疾病,是由牛21号染色体上FANCI基因3 329bp碱基缺失突变引起的,隐性基因纯合时引起胚胎早期流产或畸形,给奶牛养殖者造成很大的经济损失。本研究利用长片段PCR方法对北京地区636头母牛样品进行了检测分析,研究结果表明,在所检测的奶牛群体中,BS有害遗传缺陷基因的携带率为6.8%,有害等位基因频率为3.4%,并通过系谱追踪发现,BS遗传缺陷可追溯到美国一头非常优秀的公牛Sweet Haven Tradition(USAM1682485,BY),其后代在我国有一定的影响。通过剔除BS携带者公牛可以有效控制BS遗传缺陷的传播,但是,我国部分BS携带者公牛冻精依然在商业化使用,所以,有效监控BS携带者在奶牛群中的状况对BS剔除计划是有益的。     

荷斯坦牛脊椎畸形综合征分子诊断方法的建立与应用

脊椎畸形综合征(Complex Vertebral Malformation,CVM)是由常染色体上SLC35A3基因单碱基突变(G→T)引起的隐性遗传疾病,该基因隐性纯合(CV/CV)时奶牛致死,但CVM携带者表现正常,所以CVM携带者公牛可以通过人工授精技术传播CVM缺陷基因。本研究自行设计检测CVM特异性PCR引物,扩增长度为173bp,然后利用PCR-SSCP方法对186个公牛样品和140个母牛样品进行了检测分析,该方法简便快捷、准确率高,使用样品宽泛,适合大样本筛选。研究结果表明,在所检测的样本中,荷斯坦种公牛和母牛CVM携带率分别为11.3%和12.1%,并通过系谱追踪发现,CVM遗传缺陷的共同祖先是美国名牛"Penstate Ivanhoe Star"(USA.1441440,CV)。通过剔除CVM携带者公牛可以有效地控制CVM遗传缺陷,但是,我国许多CVM携带者公牛冻精依然在商业化使用,所以,有效地监控CVM携带者在奶牛群中的状况对CVM防控计划是有益的。     

国内荷斯坦种公牛HH4遗传缺陷基因筛查

HH4是新近在荷斯坦牛中发现的一种遗传缺陷,其分子机理为牛1号染色体上的GART基因编码区内的1个A/C错义突变。该遗传缺陷呈隐性遗传、突变等位基因纯合时,导致胚胎早期死亡。本研究针对HH4建立了基于PCR-RFLP技术的分子检测方法。结果表明:通过对466头国内荷斯坦种公牛进行基因筛查,发现6头携带者,均可以追溯到共同的祖先公牛Besne Buck。建议我国今后进口荷斯坦种牛时,对个体进行系谱分析和遗传缺陷基因分子检测,避免引入有害基因;同时,对国内现有荷斯坦公牛群体进行HH4筛查,明确标注每头公牛的携带状态,以便在生产实践中合理选种选配。     

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。     

我国荷斯坦种公牛CVM遗传缺陷基因的分子检测

研究运用引入酶切位点聚合酶链式反应(PIRA-PCR)方法检测牛脊柱畸形综合症(CVM)在我国荷斯坦种公牛中的携带情况。共检测了来自全国14个公牛站的587头种公牛,结果表明:携带者56头,携带率为9.54%。系谱分析确认40头携带者公牛是Carlin-M Ivanhoe Bell的后代。对公牛信息统计显示,2001—2007年各年度出生的种公牛都保持较高的CVM携带率(8.22%～15.56%)。美国、加拿大和澳大利亚是我国引进公牛的主要来源国,进口公牛的CVM携带率分别为11.37%、3.73%和14.29%。此外,本研究还对我国荷斯坦牛CVM遗传缺陷的控制和CVM携带公牛的利用提出建议。     

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。     

奶牛单基因遗传缺陷研究进展

综述了奶牛遗传缺陷病的危害、种类及遗传方式。对世界荷斯坦联盟和加拿大奶牛协会官方要求在奶牛上系谱注明检测结果的6种遗传缺陷病,脊椎畸形综合症、白细胞粘附缺陷症、牛尿苷酸合酶缺乏症、瓜氨酸血症、凝血因子XI缺陷症（Factor XI）、并趾症也称为骡蹄症的最新研究进展进行了概述。     

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.     

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.     

牛尿苷酸合酶缺乏症PCR-RFLP检测方法的研究

本研究通过PCR-RFLP建立了直接利用基因组DNA检测尿苷酸合酶缺乏症（DUMPS）突变的方法.对济南市6个奶牛场224头奶牛及53头荷斯坦种公牛进行了DUMPS的PCR-RFLP检测,共检出2头杂合母牛（携带者）,杂合子占检测母牛群的0.89%,在荷斯坦公牛中只检测到一种基因型,没有发现隐性突变基因的携带者.实验证明,该方法重复性好、灵敏性高、稳定性可靠.     

牛尿苷酸合酶缺乏症PCR-RFLP检测方法的建立

提取牛血液和精液DNA,进行PCR扩增、RFLP分析,建立尿苷酸合酶缺乏症(DUMPS)检测方法。对济南市6个奶牛场224头荷斯坦奶牛及53头种公牛进行了DUMPS的PCR-RFLP检测,共检测出2头杂合母牛(携带者),杂合子占检测母牛群的0.89%,在荷斯坦公牛中只检测到一种基因型,没有发现隐性突变基因的携带者。实验证明,该方法重复性好、灵敏性高、稳定性可靠。