The 80-kb DNA duplication on BTA1 is the only remaining candidate mutation for the polled phenotype of Friesian origin

Background

The absence of horns, called polled phenotype, is the favored trait in modern cattle husbandry. To date, polled cattle are obtained primarily by dehorning calves. Dehorning is a practice that raises animal welfare issues, which can be addressed by selecting for genetically hornless cattle. In the past 20 years, there have been many studies worldwide to identify unique genetic markers in complete association with the polled trait in cattle and recently, two different alleles at the POLLED locus, both resulting in the absence of horns, were reported: (1) the Celtic allele, which is responsible for the polled phenotype in most breeds and for which a single candidate mutation was detected and (2) the Friesian allele, which is responsible for the polled phenotype predominantly in the Holstein-Friesian breed and in a few other breeds, but for which five candidate mutations were identified in a 260-kb haplotype. Further studies based on genome-wide sequencing and high-density SNP (single nucleotide polymorphism) genotyping confirmed the existence of the Celtic and Friesian variants and narrowed down the causal Friesian haplotype to an interval of 145 kb.

Results

Almost 6000 animals were genetically tested for the polled trait and we detected a recombinant animal which enabled us to reduce the Friesian POLLED haplotype to a single causal mutation, namely a 80-kb duplication. Moreover, our results clearly disagree with the recently reported perfect co-segregation of the POLLED mutation and a SNP at position 1 390 292 bp on bovine chromosome 1 in the Holstein-Friesian population.

Conclusion

We conclude that the 80-kb duplication, as the only remaining variant within the shortened Friesian haplotype, represents the most likely causal mutation for the polled phenotype of Friesian origin.     

SNP-based association mapping of the polled gene in divergent cattle breeds

Naturally, hornless cattle are called polled. Although the POLL locus could be assigned to a c. 1.36‐Mb interval in the centromeric region of BTA1, the underlying genetic basis for the polled trait is still unknown. Here, an association mapping design was set up to refine the candidate region of the polled trait for subsequent high‐throughput sequencing. The case group comprised 101 homozygous polled animals from nine divergent cattle breeds, the majority represented by Galloway, Angus, Fleckvieh and Holstein Friesian. Additionally, this group included some polled individuals of Blonde d’Aquitaine, Charolais, Hereford, Jersey and Limousin breeds. The control group comprised horned Belgian Blue, Fleckvieh, Holstein Friesian and Illyrian Bu?a cattle. A genome‐wide scan using 49 163 SNPs was performed, which revealed one shared homozygous haplotype block consisting of nine neighbouring SNPs in all polled animals. This segment defines a 381‐kb interval on BTA1 that we consider to be the most likely location of the POLL mutation. Our results further demonstrate that the polled‐associated haplotype is also frequent in horned animals included in this study, and thus the haplotype as such cannot be used for population‐wide genetic testing. The actual trait‐associated haplotype may be revealed by using higher‐density SNP arrays. For the final identification of the causal mutation, we suggest high‐throughput sequencing of the entire candidate region, because the identification of functional candidate genes is difficult owing to the lack of a comparable model.     

Associations of single nucleotide polymorphisms in candidate genes with the polled trait in Datong domestic yaks

The domestic yak (Bos grunniens) is an iconic symbol of animal husbandry at high altitudes. Yaks exhibit unique external characteristics including long hair and large horns. However, hornless yaks can be found in different breeds and different populations. The hornless trait is also known as polled, and the POLL locus has been fine‐mapped to chromosome 1 in cattle (Bos taurus), although the underlying genetic basis of the polled trait is still unclear in the yak. Thus, we performed an association study to identify the genetic polymorphisms responsible for the polled trait in the yak. Fifty polled Datong domestic yaks and 51 horned individuals were selected randomly from a huge herd and were used as the case and control groups respectively for the association analysis. Twelve genes located in the candidate region of the POLL locus in cattle were used as references to detect DNA polymorphisms related to yak polledness, which were analyzed by sequencing and a high‐resolution melting test. We applied Fisher's exact test and haplotype analysis to show that a 147‐kb segment that included three protein‐coding genes C1H21orf62, GCFC1 and SYNJ1 was the most likely location of the POLL mutation in domestic yaks.     

Polledness in Argentinean Creole cattle,five centuries surviving

Polledness has been shown to have autosomal Mendelian inheritance, with the polled locus being dominant to the horned locus. This trait was mapped to the BTA1 centromeric end in several breeds. One of the distinctive attributes of Creole cattle, such as the Argentinean Creole, is the presence of long, lyre‐shaped horns. However, polled native animals were reported before the introduction of modern selected European breeds. Here, we studied the origin of the polled mutation, either independent or introgressed, in a Creole line from the Creole cattle founder group at the IIACS‐INTA Leales Experimental Station (northwest Argentina). The study sample (65 animals: 26 horned and 39 polled) was genotyped using high‐density SNP microarrays and three previously reported genetic markers (P_202ID, P_80kbID and P_G). A genome‐wide association study, selection signatures, linkage disequilibrium analysis and copy number variations were used to detect the responsible region and the segregating haplotypes/alleles. The interval mapped in the Leales herd (1.23–2.13 Mb) overlapped with the region previously reported in several European cattle breeds, suggesting that the same locus could be segregating in this population. The previously reported variants P_F and P_G were not detected, thus dismissing the Holstein‐Friesian and Nellore origins of the polled phenotype in this native breed. Conversely, the presence of the Celtic variant P_C suggests an almost complete co‐segregation. The cluster analysis rejected the hypothesis of recent introgression, which is compatible with the historical record of polled Creole cattle in northwest Argentina.     

A 2.5-Mb contig constructed from Angus, Longhorn and horned Hereford DNA spanning the polled interval on bovine chromosome 1

The polled locus has been mapped by genetic linkage analysis to the proximal region of bovine chromosome 1. As an intermediate step in our efforts to identify the polled locus and the underlying causative mutation for the polled phenotype, we have constructed a BAC-based physical map of the interval containing the polled locus. Clones containing genes and markers in the critical interval were isolated from the TAMBT (constructed from Angus and Longhorn genomic DNA) and CHORI-240 (constructed from horned Hereford genomic DNA) BAC libraries and ordered based on fingerprinting and the presence or absence of 80 STS markers. A single contig spanning 2.5 Mb was assembled. Comparison of the physical order of STSs to the corresponding region of human chromosome 21 revealed the same order of genes within the polled critical interval. This contig of overlapping BAC clones from horned and polled breeds is a useful resource for SNP discovery and characterization of positional candidate genes.     

Fine mapping of the polled locus to a 1-Mb region on bovine Chromosome 1q12

The absence of horns in Bos taurus is under genetic control of the autosomal dominant polled locus which has been genetically mapped to the centromeric region of cattle Chromosome 1. Recently a 4-Mb BAC contig of this chromosomal region has been constructed. Toward positional cloning of the bovine polled locus, we identified 20 additional microsatellite markers spread over the contig map by random sequencing of bacterial artificial chromosome (BAC) subclones. A total of 26 markers were genotyped in 30 two-generation half-sib families of six different German cattle breeds segregating for the hornless phenotype including 336 informative meioses for the polled character. Our fine-mapping study involving 19 recombinant haplotypes allowed us to narrow the critical region for the bovine polled locus to a 1-Mb segment with a centromeric boundary at RP42-218J17_MS1 and a telomeric boundary at BM6438. For marker-assisted selection purposes, the first evidence of informative flanking markers helps to predict polled genotypes with a higher degree of accuracy within families until testing of the causative mutation is available.     

SNP-based association mapping of Arachnomelia in Fleckvieh cattle

Bovine arachnomelia is an inherited congenital disorder with malformation mainly of the limbs, the vertebral column and the skull, following a monogenic autosomal recessive heredity. Despite almost identical pathological findings, arachnomelia has previously been mapped to bovine chromosome 23 and 5 in Fleckvieh and Braunvieh respectively. Therefore, this disorder may be an example of locus heterogeneity in cattle. This study aimed to refine the candidate region to allow positional cloning and sequence analyses of candidate genes in Fleckvieh cattle. For that purpose, a case-control association mapping design was set up with a case group of 16 pre-selected affected individuals and a control group of 50 unrelated animals. The subset of affected animals was selected from a total of 129 pathologically confirmed cases due to the occurrence of recombination(s) within a 14.5 cM candidate interval previously mapped to chromosome 23. Six linked microsatellites currently used for indirect gene testing in Fleckvieh were analysed for this purpose. In all selected cases, a genome-wide scan using 44 473 informative SNPs revealed shared segments of homozygosity at 15 adjacent SNPs on chromosome 23. Additional haplotype analysis of 37 carrier bulls confirmed the localization of the arachnomelia locus to a region of 927 kb (13.622-14.549 Mb) containing molybdenum cofactor biosynthesis protein 1 gene, the most likely candidate gene for arachnomelia in Fleckvieh. The number of recombinant haplotypes observed in cases was more than doubled compared with the number of expected recombinations. This remarkably increased mapping resolution and thus illustrates the benefit of pre-selection in association studies.     

Assignment of the locus for arachnomelia syndrome to bovine chromosome 23 in Simmental cattle

Arachnomelia syndrome is a lethal inherited malformation mainly of the limbs, vertebral column and skull in cattle, which poses a severe impairment to farmers and breeders. Recently, a number of cases of arachnomelia syndrome have occurred in the Simmental breed and some sires with excellent breeding values had been shown to be carriers of the disease. We herein report the genetic mapping of the mutation underlying arachnomelia in cattle. The disease was mapped using a two-stage genome scan. A first round autosomal genome-wide screening using a limited number of cases identified three chromosomal regions with lod-scores > 1. The position of the arachnomelia syndrome locus was identified to be on BTA 23 by genotyping an additional, independent set of animals with markers that provided positive lod-scores in the course of the initial genome-wide screen. Using a denser set of regional microsatellites, the locus could be mapped to a region about 9 cM in length. The most significant linkage signal with arachnomelia syndrome was obtained with marker NRKM-17 (lod-score > 20) using a recessive model. Interestingly, different genes seem to be responsible for the disease in Brown Swiss and Simmental breeds, as arachnomelia syndrome was mapped to a different location in Brown Swiss. The results provide sufficient information for the development of a genetic test system and also allow the identification of positional candidate genes.     

Fine‐mapping the POLL locus in Brahman cattle yields the diagnostic marker CSAFG29

The POLL locus has been mapped to the centromeric region of bovine chromosome 1 (BTA1) in both taurine breeds and taurine–indicine crosses in an interval of approximately 1 Mb. It has not yet been mapped in pure‐bred zebu cattle. Despite several efforts, neither causative mutations in candidate genes nor a singular diagnostic DNA marker has been identified. In this study, we genotyped a total of 68 Brahman cattle and 20 Hereford cattle informative for the POLL locus for 33 DNA microsatellites, 16 of which we identified de novo from the bovine genome sequence, mapping the POLL locus to the region of the genes IFNAR2 and SYNJ1. The 303‐bp allele of the new microsatellite, CSAFG29, showed strong association with the POLL allele. We then genotyped 855 Brahman cattle for CSAFG29 and confirmed the association between the 303‐bp allele and POLL. To determine whether the same association was found in taurine breeds, we genotyped 334 animals of the Angus, Hereford and Limousin breeds and 376 animals of the Brangus, Droughtmaster and Santa Gertrudis composite taurine–zebu breeds. The association between the 303‐bp allele and POLL was confirmed in these breeds; however, an additional allele (305 bp) was also associated but not fully predictive of POLL. Across the data, CSAFG29 was in sufficient linkage disequilibrium to the POLL allele in Australian Brahman cattle that it could potentially be used as a diagnostic marker in that breed, but this may not be the case in other breeds. Further, we provide confirmatory evidence that the scur phenotype generally occurs in animals that are heterozygous for the POLL allele.     

Estimation of DNA sequence diversity in bovine cytokine genes

DNA sequence variation provides the fundamental material for improving livestock through selection. In cattle, single nucleotide polymorphisms and small insertions/deletions (collectively referred to here as SNPs) have been identified in cytokine genes and scored in a reference population to determine linkage map positions. The aim of the present study was twofold: first, to estimate the SNP frequency in a reference population of beef cattle, and second, to determine cytokine haplotypes in a group of sires from commercial populations. Forty-five SNP markers in DNA segments from nine cytokine gene loci were analyzed in 26 reference parents. Comparison of all 52 haploid genomes at each PCR amplicon locus revealed an average of one SNP per 143 bp of sequence, whereas comparison of any two chromosomes identified heterozygous sites, on average, every 443 bp. The combination of these 45 SNP genotypes was sufficient to uniquely identify each of the 26 animals. The average number of haplotype alleles (4.4) per PCR amplicon (688 bp) and the percentage heterozygosity among founding parents (50%) were similar to those for microsatellite markers in the same population. For 49 sires from seven common breeds of beef cattle, SNP genotypes (1225 total) were obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) at three amplicon loci. All three of the amplicon haplotypes were correctly deduced for each sire without the use of parent or progeny genotypes. The latter allows a wide range of genetic studies in commercial populations of cattle where genotypic information from relatives may not be available. Received: 16 June 2000 / Accepted: 23 August 2000     

Fine genetic localization of the gene for autosomal dominant polycystic kidney disease (PKD1) with respect to physically mapped markers.

PKD1, the gene for the chromosome 16-linked form of autosomal dominant polycystic kidney disease, has previously been genetically mapped to an interval bounded by the polymorphic loci Fr3-42/EKMDA2 distally and O327hb/O90a proximally. More recently, 26.6PROX was identified as the closest proximal flanking locus. We set out to refine the localization of PKD1 by identifying a series of single recombinant events between the flanking markers Fr3-42/EKMDA2 and O327hb/O90a and analyzing them with a new set of polymorphic loci that have been physically mapped within the PKD1 interval. We identified 11 such crossovers in eight families; 6 of these fell into the interval between GGG1 and 26.6PROX, a distance of less than 750 kb. Three of these crossovers placed PKD1 proximal to GGG1 and two crossovers placed PKD1 distal to 26.6PROX. Both of the latter also placed PKD1 telomeric to a locus 92.6SH1.0, which lies 200-250 kb distal to 26.6PROX. The sixth recombinant, however, placed the disease mutation proximal to the locus 92.6SH1.0. Several possible explanations for these observations are discussed. An intensive study to locate deletions, insertions, and other chromosomal rearrangements associated with PKD1 mutations failed to detect any such abnormalities. Thus we have defined, in genetic and physical terms, the segment of 16p13.3 where PKD1 resides and conclude that a gene-by-gene analysis of the region will be necessary to identify the mutation(s).     

Populationwide Investigation of Two Indel Polymorphisms at the Prion Protein Gene in Polish Holstein�CFriesian Cattle

The allele, genotype, and haplotype frequencies among 837 Polish Holstein-Friesian cattle were determined at two regulatory indel polymorphisms of the PRNP gene. Allele frequencies at the 23 bp indel promoter polymorphism were 0.622 (del) and 0.378 (ins), with 0.613 and 0.387 in sires and 0.633 and 0.366 in dams. Allele frequencies at the 12 bp indel intron polymorphism were 0.527 (del) and 0.473 (ins), with 0.529 and 0.471 in sires and 0.543 and 0.456 in dams. Four haplotypes were identified in this population (23-12del, 23-12ins, 23del-12ins, and 23ins-12del). Haplotype 23-12del occurred most frequently in both sire and dam groups. Comparative analysis of Polish Holstein-Friesian and German Holstein populations revealed a similar genetic structure for the 23 bp indel polymorphism and a significantly different one for the 12 bp indel polymorphism. In allele and haplotype analysis, significant differences were observed between the Polish Holstein-Friesian population and a BSE-free German Holstein population.     

Localization of psoriasis-susceptibility locus PSORS1 to a 60-kb interval telomeric to HLA-C

Recent genome scans have established the presence of a major psoriasis-susceptibility locus in the human leukocyte antigen (HLA) complex on chromosome 6p21.3. To narrow the interval for candidate gene testing, we performed a linkage-disequilibrium analysis of 339 families, with the use of 62 physically mapped microsatellite markers spanning the major histocompatibility complex (MHC). As detected by use of the transmission/disequilibrium test (TDT), individual markers yielded significant linkage disequilibrium across most of the MHC. However, the strongest evidence for marker-trait disequilibrium was found in an approximately 300-kb region extending from the MICA gene to the corneodesmosin gene. Maximum-likelihood haplotypes were constructed across the entire MHC in the original sample and across a 1.2-Mb region of the central MHC in an expanded sample containing 139 additional families. Short (two- to five-marker) haplotypes were subjected to the TDT using a "moving-window" strategy that reduced the variability of TDT P values relative to the single-locus results. Furthermore, the expanded sample yielded a sharp peak of evidence for linkage disequilibrium that spanned approximately 170 kb and that was centered 100 kb telomeric to HLA-C. The 1.2-Mb interval was further dissected by means of recombinant ancestral haplotype analysis. This analysis identified risk haplotype 1 (RH1), which is a 60-kb fragment of ancestral haplotype 57.1, on all identifiable HLA risk haplotypes. One of these haplotypes exhibits significant linkage disequilibrium with psoriasis but does not carry Cw6, which is the HLA allele most strongly associated with the disease. These results demonstrate that RH1 is highly likely to carry the disease allele at PSORS1, and they exclude HLA-C and corneodesmosin with a high degree of confidence.     

Genetic mapping of the belt pattern in Brown Swiss cattle to BTA3

The white belt pattern of Brown Swiss cattle is characterized by a lack of melanocytes in a stretch of skin around the midsection. This pattern is of variable width and sometimes the belt does not fully circle the body. To identify the gene responsible for this colour variation, we performed linkage mapping of the belted locus using six segregating half-sib families including 104 informative meioses for the belted character. The pedigree confirmed a monogenic autosomal dominant inheritance of the belted phenotype in Brown Swiss cattle. We performed a genome scan using 186 microsatellite markers in a subset of 88 animals of the six families. Linkage with the belt phenotype was detected at the telomeric region of BTA3. Fine-mapping and haplotype analysis using 19 additional markers in this region refined the critical region of the belted locus to a 922-kb interval on BTA3. As the corresponding human and mouse chromosome segments contain no obvious candidate gene for this coat colour trait, the mutation causing the belt pattern in the Brown Swiss cattle might help to identify an unknown gene influencing skin pigmentation.     

Linkage disequilibrium mapping in domestic dog breeds narrows the progressive rod-cone degeneration interval and identifies ancestral disease-transmitting chromosome

Canine progressive rod-cone degeneration (prcd) is a retinal disease previously mapped to a broad, gene-rich centromeric region of canine chromosome 9. As allelic disorders are present in multiple breeds, we used linkage disequilibrium (LD) to narrow the approximately 6.4-Mb interval candidate region. Multiple dog breeds, each representing genetically isolated populations, were typed for SNPs and other polymorphisms identified from BACs. The candidate region was initially localized to a 1.5-Mb zero recombination interval between growth factor receptor-bound protein 2 (GRB2) and SEC14-like 1 (SEC14L). A fine-scale haplotype of the region was developed, which reduced the LD interval to 106 kb and identified a conserved haplotype of 98 polymorphisms present in all prcd-affected chromosomes from 14 different dog breeds. The findings strongly suggest that a common ancestor transmitted the prcd disease allele to many of the modern dog breeds and demonstrate the power of the LD approach in the canine model.     

A new locus for autosomal dominant amelogenesis imperfecta on chromosome 8q24.3

Amelogenesis imperfecta (AI) is a collective term used to describe phenotypically diverse forms of defective tooth enamel development. AI has been reported to exhibit a variety of inheritance patterns, and several loci have been identified that are associated with AI. We have performed a genome-wide scan in a large Brazilian family segregating an autosomal dominant form of AI and mapped a novel locus to 8q24.3. A maximum multipoint LOD score of 7.5 was obtained at marker D8S2334 (146,101,309 bp). The disease locus lies in a 1.9 cM (2.1 Mb) region according to the Rutgers Combined Linkage-Physical map, between a VNTR marker (at 143,988,705 bp) and the telomere (146,274,826 bp). Ten candidate genes were identified based on gene ontology and microarray-facilitated gene selection using the expression of murine orthologues in dental tissue, and examined for the presence of a mutation. However, no causative mutation was identified.