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.     

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.     

5个黄牛群体垂体转录因子基因变异研究

 为了阐明云南黄牛垂体转录因子(POU1F1)基因的群体变异特征,采用DNA序列分析和PCR-RFLP技术对云南地区5个黄牛群体（3个本地群体和2个引进品种）的垂体转录因子(POU1F1)基因的第5内含子和第6外显子进行了基因克隆测序和群体变异检测分析。在5个群体中均发现存在A和B两个等位基因,在国外引进的短角牛和安格斯牛中,A等位基因为优势等位基因,其基因频率分别为0.944和0.700;而在云南的昭通黄牛、迪庆黄牛和荷斯坦奶牛中B等位基因占优势,其基因频率分别为0.645,0.727和0.917;在短角牛和安格斯牛中,BB基因型频率为0;而在云南荷斯坦奶牛中,AA基因型频率为0。在所检测的座位中,昭通黄牛、迪庆黄牛和安格斯牛具有较高的杂合度,而其他群体该座位杂合度较低。     

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.     

Genetic diversity among some cattle breeds in the Alpine area

Seven autochthonous Italian cattle breeds bred in the alpine area (Aosta Black Pied, Aosta Red Pied, Aosta Chestnut, Oropa Red Pied, Grey Alpine, Rendena and Burlina) were investigated in order to characterise their genetic structure and to study their phylogenetic origin. Two cattle breeds from Germany (Original German Brown and Holstein) and four from Switzerland (Simmental, Herens, Evolene and Brown Swiss) were included in the study in order to determine the genetic diversity existing among Italian local breeds, similar breeds bred on the other side of the Alps and in the well known Holstein.
Seventeen microsatellites, of the internationally accepted panel for the study of cattle biodiversity, were used for the analysis.
Microsatellites were highly polymorphic with a mean number of 5,5 alleles (ranging from 2 to 12 per locus). For each locus, allelic frequencies, heterozygosity (H) and the Polymorphism Information Content (PIC) were computed. The genetic equilibrium according to Hardy–Weinberg was calculated for each population and for each locus. Allele frequencies were used to estimate genetic distances and to draw a phylogenetic tree. The two closest breeds were Aosta Red Pied and Aosta Black Pied, while the two genetically most different were Holstein and Aosta Chestnut. Aosta valley breeds, Evolene and Herens constituted a tight cluster in the phylogenetic consensus tree. Principal component analysis showed a similar pattern for all the alpine breeds, while Holstein and Original German Brown were far away. The genetic differences among breeds were in accordance with their geographical and historical origins.     

8个牛品种的大理石花纹评分相关基因变异的微卫星DNA池分析

本研究以8个牛品种为研究对象,利用与大理石花纹评分基因相关的7个微卫星位点结合DNA池分析技术,探讨微卫星DNA多态性与8个牛品种大理石花纹评分间的关系,并根据性状同质性原理预测品种组合,以加快生产出高端"雪花"牛肉。结果表明:日本和牛与荷斯坦牛、安格斯牛、渤海黑牛的相似性系数均超过0.8,而日本和牛与其他牛品种的的相似性系数均小于0.8。利用MEGA4软件采用邻接法进行聚类,利木赞牛与西门塔尔牛先聚合在一起,再与草原红牛聚合;渤海黑牛与鲁西黄牛2个地方良种聚合在一起,以上5个品种聚为第1大类。荷斯坦牛与日本和牛聚合,再与安格斯牛聚在一起,它们聚为第2大类。根据本研究结果和国外肉牛杂交生产实践,从牛肉大理石花纹性状的同质性出发,建议利用日本和牛、荷斯坦牛为亲本,杂交生产高档"雪花"牛肉,以解决我国的肉牛牛源短缺问题。     

Genetic and phenotypic parameters for conformation and yield traits in three Swiss dairy cattle breeds

This study presents genetic parameters for conformation traits and their genetic and phenotypic correlations with milk production traits and somatic cell score (SCS) in three Swiss dairy cattle breeds. Data on first lactations from Holstein (67 839), Brown Swiss (173 372) and Red & White breeds (53 784) were available. Analysed conformation traits were stature and heart girth (both in cm), and linear scores of body depth, rump width, dairy character or muscularity, and body condition score (only in Holstein). A sire model, with relationships among sires, was used for all breeds and traits and variance components were estimated using AS‐REML. Heritabilities for stature were high (0.6–0.8), and for the linear type traits ranged from 0.3 to 0.5, for all breeds. Genetic correlations with production traits (milk, fat and protein yield) and SCS differed between the dairy breeds. Most markedly, stronger correlations were found between SCS and some conformation traits in Brown Swiss and Red & White, indicating that a focus on a larger and more ‘dairy’ type in these breeds would lead to increased SCS. Another marked difference was that rump width correlated positively with milk yield traits in Holstein and Red & White, but negative in Brown Swiss. Results indicate that conformation traits generally can be used as predictors for various purposes in dairy cattle breeding, but may require specific adaptation for each breed.     

Identification of differential selection traces in two Polish cattle breeds

Genetic improvement of animals based on artificial selection is leading to changes in the frequency of genes related to desirable production traits. The changes are reflected by the neutral, intergenic single nucleotide polymorphims (SNPs) being in long‐range linkage disequilibrium with functional polymorphisms. Genome‐wide SNP analysis tools designed for cattle, allow for scanning divergences in allelic frequencies between distinct breeds and thus for identification of genomic regions which were divergently selected in breeds' histories. In this study, by using Bovine SNP50 assay, we attempted to identify genomic regions showing the highest differences in allele frequencies between two distinct cattle breeds – preserved, unselected Polish Red breed and highly selected Holstein cattle. Our study revealed 19 genomic regions encompassing 55 protein‐coding genes and numerous quantitative trait loci which potentially may underlie some of the phenotypic traits distinguishing the breeds.     

Bovine Leukocyte Adhesion Deficiency in Danish Holstein-Friesian Cattle: I. PCR Screening and Allele Frequency Estimation

Jergensen, C. B., J. S. Agerholm, J. Pedersen and P. D. Thomsen: Bovine leukocyte adhesion deficiency in Danish Holstein-Friesian Cattle I. PCR screening and allele frequency estimation. Acta vet. Scand. 1993,34,231-236.–A screening program for bovine leukocyte adhesion deficiency (BLAD) in Danish Holstein-Friesian cattle has been initiated. During the first months 1611 animals were tested by a PCR based assay. Of these animals 1256, 346, and 8 were assigned normal, BLAD carriers, and BLAD affected animals, respectively One bull, born as a co-twin, showed weak reaction for the BLAD allele on DNA isolated from leukocytes, but a normal genotype on DNA isolated from semen. Chromosome analysis showed that this bull was a blood chimaera. Estimation of the BLAD allele frequency upon the PCR test results showed that around 450 Danish calves born in 1991 might have been affected with the recessive disorder.     

中国荷斯坦牛白细胞粘附缺陷病遗传分析

荷斯坦牛白细胞粘附缺陷病（BLAD）是一种遗传性免疫缺陷病，患病牛出生后，生长发育差，绝大多数在1年内死亡，且不具繁殖和哺育能力。该病的遗传基础为CD18基因编码区383位的A／G点突变导致白细胞表面的β2整合素表达缺陷。目前欧美等奶牛业发达国家都已经建立了完善的BLAD分子检测方法和跟踪监控体系。中国长期从国外进口荷斯坦公牛精液、胚胎或活体，由此可能引进了BLAD基因。本研究采用PCR—RFLP方法对116头可疑中国荷斯坦牛进行了检测．确定了2头BLAD携带者公牛和8头携带者母牛．未发现隐性纯合个体．     

Sequence variation of bovine prion protein gene in Japanese cattle (Holstein and Japanese Black)

To assess relationships between nucleotide polymorphisms of the prion protein (PRNP) gene and susceptibility to bovine spongiform encephalopathy (BSE), we investigated polymorphisms in the open reading frame (ORF) and 2 upper regions of the PRNP gene from 2 Japanese cattle breeds: 863 healthy Holstein cattle, 6 BSE-affected Holstein cattle, and 186 healthy Japanese Black (JB) cattle. In the ORF, we found single-nucleotide polymorphisms (SNPs) at nucleotide positions 234 and 576 and found 5 or 6 copies of the octapeptide repeat, but we did not find any amino acid substitutions. In the upper region, we examined 2 sites of insertion/deletion (indel) polymorphisms: a 23-bp indel in the upper region of exon 1, and a 12-bp indel in the putative promoter region of intron 1. A previous report suggests that the 23-bp indel polymorphism is associated with susceptibility to BSE, but we did not find a difference in allele frequency between healthy and BSE-affected Holstein cattle. There were differences in allele frequency between healthy Holstein and JB cattle at the 23- and 12-bp indels and at the SNPs at nucleotide positions 234 and 576, but there was no difference in allele frequency of the octapeptide repeat. We identified a unique PRNP gene lacking a 288-bp segment (96 amino acids) in DNA samples stocked in our laboratory, but this deletion was not found in any of the 1049 cattle examined in the present study. The present results provide data about variations and distribution of the bovine PRNP gene.     

Genetic differences among native and modern cattle breeds in Lithuania based on milk protein loci polymorphism

The intrabreed and interbreed genetic diversity of Lithuanian cattle breeds - two native, namely Lithuanian Light Grey and Lithuanian White-Backed and two modern, namely Lithuanian Red and Lithuanian Black and White was investigated by determination of genetic markers: 4 milk protein systems, Alpha(s1)-casein, Kappa-casein, Beta-casein and Beta-lactoglobulin, which are comprised of 12 different milk protein types. According to results, the B type of Alpha(s1)-casein was found as predominant in all four studied breeds. The most common A and B types of Kappa-casein were found at high frequency in all investigated cattle breeds. All investigated Lithuanian dairy cattle breeds had high frequency of Beta-lactoglobulin whey protein B types, with the highest frequency in Lithuanian Red breed, and the lowest in Lithuanian Light Grey. After investigation the diversity of alleles and genotypes of milk proteins in Lithuanian dairy cattle breeds was determined that, Lithuanian Red breed was distinguished private C allele and BC genotype of Beta-lactoglobulin and CC genotype of Alpha(s1)-casein. The interbreed genetic diversity was estimated by a principal component analysis (PCA). The first principal component (PC) explains 63.39% and the second principal component (PC) explains 33.67% of the genetics diversity between the breeds. Principal component analysis, suggests the hypothesis that native Lithuanian White Backed and Lithuanian Light Grey breeds still have traits tracing to old native populations.     

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.     

The T allele at the g.1471620G>T in the EDG1 gene associated with high marbling in Japanese Black cattle is at a low frequency in breeds not selected for marbling

Our previous study detected a single nucleotide polymorphism (SNP), g.1471620G > T , in the 5' flanking region of the endothelial differentiation sphingolipid G-protein-coupled receptor 1 ( EDG1 ) gene, which has been considered as a positional functional candidate for the gene responsible for marbling, and showed association of the g.1471620G > T SNP with marbling in Japanese Black beef cattle. In the present study, we investigated the allele frequency distribution of the g.1471620G > T SNP among the 5 cattle breeds, Japanese Black, Japanese Brown, Japanese Short Horn, Holstein, and Brown Swiss breeds. The T allele at the g.1471620G > T SNP associated with high marbling was found at high frequency in Japanese Black breed that has been subjected to a strong selection for high marbling, while the allele was absent or at very low frequencies in the other breeds that have not been strongly selected for high marbling. Based on this finding, we hypothesized that the pressure of the strong selection for high marbling in Japanese Black breed has increased the frequency of the T allele at the g.1471620G > T SNP in the EDG1 .     

Correlations Analysis between Polymorphism of 3’-UTR in MSTN Gene and Slaughter Traits of China Red Steppe

In order to study the effect of myostatin (MSTN) gene on the meat performance of Local Yellow cattle in China,selecting 2 Local cattle breeds (Grassland Red bull,Yanbian cattle) as the research object,four binary hybrid cattle group (Germany Yellow cattle×Simmental,Limousin×Simmental,Charolais×Simmental and Red Angus×Simmental) as reference.PCR-RFLP and DNA sequencing methods were conducted to detect single nucleotide polymorphism of MSTN gene in two Chinese native cattle and four cross cattle,and analyzed the correlations between single nucleotide polymorphism of China Red Steppe MSTN gene and slaughter traits.The sequence analysis showed that there was no mutations in the MSTN gene exon 3,a C5358A mutation of 3'-UTR of MSTN gene was found,which made the AclⅠrestriction enzyme site disappeared,all cattle groups showed polymorphism which was formed two genes as A,C, and 3 genotypes as AA,CA and CC, respectively.The research results suggested that AA genotype in groups of four hybrid cattle had dominant advantage and the genotype frequency was apparently higher than two groups of Chinese Native cattle for the test of independence,allele A was expressed the preferential allelic,the frequencies of CA,CC and AA genotypes showed a little variation between two cross cattle and Native cattle,and the frequency of allele C was apparently higher than allele A,so allele C was designed the preferential allelic in the local population.While correlations between different genotypes of China Red Steppe and slaughter traits analysis showed that AA genotype individual net meat percentage and eye muscle area were significantly higher than CA and CC genotype individuals (P<0.05),but other slaughter straits had no significant differences in three genotypes(P>0.05).These results provided a useful reference for research of the meat performance of China Red Steppe in the molecular breeding field.     

隆林牛与郏县红牛线粒体DNA全基因组遗传多样性比较研究

摘　要：［目的］本研究旨在从基因组水平探究隆林牛和郏县红牛的线粒体DNA（mtDNA）全基因组遗传多样性与母系起源,并对2个黄牛品种的mtDNA全基因组遗传多样性进行比较分析。［方法］采用全基因组重测序及生物信息学方法。［结果］在15头隆林牛和28头郏县红牛mtDNA全基因组序列中,共检测到36种单倍型,其中郏县红牛有26种单倍型,隆林牛仅有8种单倍型,2个黄牛品种共享2种单倍型。郏县红牛和隆林牛的平均单倍型多样度（Hd）分别为1.000和0.943,平均核苷酸多样度（Pi）分别为0.0080和0.0053,表明其遗传多样性丰富。构建的系统发育树表明,隆林牛和郏县红牛具有瘤牛和普通牛两个母系支系。［结论］隆林牛以瘤牛起源为主,郏县红牛为普通牛与瘤牛的混合起源,这2个地方黄牛品种具有独特的母系遗传信息,表现出明显的母系遗传差异。     

牛羊多胎性状的分子遗传基础研究

本研究采用了RAPD、PCR RFLP、微卫星、PCR SSCP、序列分析等方法对2个牛品种(秦川牛和荷斯坦奶牛共60头)、6个中国固有绵羊品种(多胎品种小尾寒羊,双胎品种大尾寒羊,单胎品种兰州大尾羊、蒙古羊、同羊和哈萨克羊共197余只`进行了分子遗传基础研究,旨在寻找牛羊多胎性状合适的分子标记,为进一步对牛双胎基因、绵羊多胎基因的探索和高繁殖率牛羊的选育提供科学依据.     

Mitochondrial DNA diversity of Anatolian indigenous domestic goats

Anatolia has been an important region for civilizations and agricultural revolution as a major domestication centre for livestock species. Goats (Capra hircus) were among the earliest domesticated animals in this region. In this study, genetic diversity of Anatolian goat breeds was characterized by comparison of mitochondrial DNA hypervariable region 1. A total of 295 individuals, including 99 Anatolian Black goats, 96 Angora goats and 100 Kilis goats, were used. Haplogroup A was found to be the dominant haplogroup in all three breeds. The highest haplogroup diversity, including haplogroups A, B2, C and G, was observed in the Anatolian Black breed. Haplogroup D was only observed in Kilis and Angora goats. Haplogroup G was found in Angora and Anatolian Black breeds. The Anatolian goat breeds had high genetic diversity values and a weak phylogeographical structure. The nucleotide diversity values were found to be higher than those in previously studied goat breeds. The fact that Anatolia is a domestication centre and its geographical position as a junction of trade routes may have caused the higher genetic diversity of Anatolian goat breeds.