Revisiting the quantitative trait loci for milk production traits on BTA6.

A parallel association study was performed in two independent cattle populations based on 41 validated, targeted single nucleotide polymorphisms (SNPs) and four microsatellite markers to re-evaluate the multiple quantitative trait loci (QTL) architecture for milk performance on bovine chromosome 6 (BTA6). Two distinct QTL located in the vicinity of the middle region of BTA6, but differing unambiguously regarding their effects on milk composition and yield traits were validated in the German Holstein population. A highly significant association of the protein variant ABCG2 p.Tyr581Ser with milk composition traits reconfirmed the causative molecular relevance of the ABCG2 gene in QTL region 1, whereas in QTL region 2, significant and tentative associations between gene variants RW070 and RW023 (located in the promoter region and exon 9 of the PPARGC1A gene for milk yield traits) were detected. For the German Fleckvieh population, only RW023 showed a tentative association with milk yield traits, whereas those loci with significant effects in German Holsteins (ABCG2 p.Tyr581Ser, RW070) showed fixed alleles. Even though our new data highlight two variants in the PPARGC1A gene (RW023, RW070) in QTL region 2, based on the results of our study, currently no unequivocal conclusion about the causal background of this QTL affecting milk yield traits can be drawn. Notably, the German Holstein and Fleckvieh populations, known for their divergent degree of dairy type, differ substantially in their allele frequencies for the growth-associated NCAPG p.Ile442Met locus.

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.

The evolution and maintenance of polymorphism in the major histocompatibility complex.

Lambs with the G2 allele at the ovine major histocompatibility complex (mhc) class II locus DRB1 has previously been shown to have lower faecal nematode egg counts than lambs with the I allele at this locus. This association has been confirmed in separate cohorts from the same farm. Other alleles within the mhc have also shown associations with nematode resistance in other breeds of sheep. Therefore, variation in the mhc is responsible for part of the observed genetic variation in resistance to nematode infection. In addition to the specific effect of particular alleles, heterozygotes are also more resistant than homozygotes. This heterozygote advantage is capable of maintaining the high levels of polymorphism observed within the mhc.

Detection and characterization of SNPs useful for identity control and parentage testing in major European dairy breeds.

We propose the use of single nucleotide polymorphisms (SNPs) instead of polymorphic microsatellite markers for individual identification and parentage control in cattle. To this end, we present an initial set of 37 SNP markers together with a gender-specific SNP for identity control and parentage testing in the Holstein, Fleckvieh and Braunvieh breeds. To obtain suitable SNPs, a total of 91.13 kb of random genomic DNA was screened yielding 531 SNPs. These, and 43 previously identified SNPs, were subjected to the following selection criteria: (1) the frequency of the minor allele must be larger than 0.1 in at least two of the three examined breeds, and (2) markers should not be linked closely. Allele frequencies were estimated by analysing sequencing traces of pooled DNA or by genotyping individual DNA samples. The selected SNP loci were physically mapped by radiation hybrid mapping or by fluorescence in situ hybridization, and tested against the neutral mutation hypothesis. The presented marker set theoretically allows probabilities of identity less than 10(-13) for individual verification and exclusion powers exceeding 99.99% for parentage testing.

An enhanced linkage map of the sheep genome comprising more than 1000 loci.

A medium-density linkage map of the ovine genome has been developed. Marker data for 550 new loci were generated and merged with the previous sheep linkage map. The new map comprises 1093 markers representing 1062 unique loci (941 anonymous loci, 121 genes) and spans 3500 cM (sex-averaged) for the autosomes and 132 cM (female) on the X chromosome. There is an average spacing of 3.4 cM between autosomal loci and 8.3 cM between highly polymorphic [polymorphic information content (PIC) > or = 0.7] autosomal loci. The largest gap between markers is 32.5 cM, and the number of gaps of > 20 cM between loci, or regions where loci are missing from chromosome ends, has been reduced from 40 in the previous map to 6. Five hundred and seventy-three of the loci can be ordered on a framework map with odds of > 1000 : 1. The sheep linkage map contains strong links to both the cattle and goat maps. Five hundred and seventy-two of the loci positioned on the sheep linkage map have also been mapped by linkage analysis in cattle, and 209 of the loci mapped on the sheep linkage map have also been placed on the goat linkage map. Inspection of ruminant linkage maps indicates that the genomic coverage by the current sheep linkage map is comparable to that of the available cattle maps. The sheep map provides a valuable resource to the international sheep, cattle, and goat gene mapping community.

Assessment of the nucleotide sequence variability in the bovine T-cell receptor alpha delta joining gene region.

The sequence of 2,193 nucleotides from the bovine T-cell receptor alpha/delta joining gene region (TCRADJ) was determined and compared with the corresponding human and murine sequences. The identity was 75.3% for the comparison of the Bos taurus vs. the Homo sapiens sequence and 63.8% for the Bos taurus vs. the Mus musculus sequence. This comparison permitted the identification of the putatively functional elements within the bovine sequence. Direct sequencing of 2,110 nucleotides in nine animals revealed 12 variable sites. Estimates, based on direct sequencing in three Holstein Friesian animals, for the two measures of sequence variability, nucleotide polymorphism (u) and nucleotide diversity (p), were 0.00050 (60.00036) and 0.00077 (60.00056), respectively. The test statistic, Tajima's D, for the comparison of the two measures indicates that the difference between u and p is close to significance (P < 0.05), suggesting the possibility of selective forces acting on the studied genomic region. Allelic variation at 5 of the 12 variable sites was analysed in 359 animals (48 Anatolian Black, 56 Braunvieh, 115 Fleckvieh, 47 Holstein Friesian, 50 Simmental and 43 Pinzgauer) using the oligonucleotide ligation assay (OLA) in combination with the enzyme linked immunoabsorbant assay (ELISA). Nine unambiguous haplotypes could be derived based on animals with a maximum of one heterozygous site. Four to seven haplotypes were present in the different breeds. When taking into account the frequencies of the haplotypes in the different breeds, especially in Anatolian Black, an ancestral cattle population, we could establish the likely phylogenetic relationships of the haplotypes. Such haplotype trees are the basis for cladistic candidate gene analysis. Our study demonstrates that the systematic search of single nucleotide polymorphisms (SNPs) is useful for analysing all aspects of variability of a given genomic region.

Construction and characterization of a gridded cattle BAC library.

A bovine genomic large-insert bacterial artificial chromosome (BAC) library has been constructed from leukocytes of a Holstein-Friesian male. Size fractionated DpnII-digested genomic DNA was ligated to the dephosphorylated BamH1 ends of a pBACe3.6 vector. Approximately 8.3 x 10(4) individual BAC clones were picked into 384-well plates. Two-hundred and sixty-seven randomly chosen clones were characterized by pulsed-field gel electrophoresis (PFGE). The average insert size was 104 kb with a frequency of clones without inserts of 5.5%. Thirty-four BAC clones were mapped by fluorescence in situ hybridization (FISH) to cattle chromosomes. Three showed signals at more than one location, one of them on the centromeric regions of all autosomes, indicating that the clone contains centromeric repeats. A subset of these BAC clones was used for the development of sequence tagged sites. Both subcloning and direct sequencing of the BACs were used for generating sequence tagged site information. The clones from the library were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones. Membranes and superpools are available through the Resource Centre of the German Human Genome Project in Berlin (http:// www.rzpd.de).

The cattle interleukin-13 gene: genomic organization, chromosomal location, and evolution of the promoter.

Interleukin (IL)-13 plays a key role in the T-helper 2 type immune response. In domestic animal species no sequence information is available for the IL13 gene. Here, we report the isolation and sequencing of the cattle IL13 gene. Since a heterologous cDNA probe failed to hybridize to cattle genomic DNA the gene was identified by a positional sequencing approach. In human and mouse both cytokines are located within a region of about 30 kilobases (kb). Therefore the region upstream of the cattle IL4 gene has been sequenced by a shotgun approach. The cattle IL13 gene was found to be located about 20 kb upstream of IL4 in a head-to-tail orientation. The exon-intron structure and the promoter region is well conserved across species. The alignment of the IL13 sequences from different species allowed the identification of highly conserved amino acid positions and transcriptional elements. Two conserved amino acid positions (glu(31) and pro(94)) are most probably homologous to the interleukin-4 signalling residues. One of the potential binding sites for transcription factors is located at position -197 to -189 and is closely related to the P elements of the IL4 promoter. Nevertheless, the overall identity of the promoter sequences of IL13 and IL4 is low. Therefore we conclude that both cytokines use diverse regulatory elements. There is evidence that IL-13, but not IL-14, is the key mediator in allergic asthma. Furthermore IL-13 is involved in resistance to some infections. Therefore IL-13 is of interest for disease association studies in domestic animals.

A 5x genome coverage bovine BAC library: production, characterization, and distribution.

A bovine large-insert DNA library has been constructed in a Bacterial Artificial Chromosome (BAC) vector. The source DNA was derived from lymphocytes of a Jersey male. High-molecular-weight DNA fragments were produced by treatment with EcoRI/EcoRI methylase and cloned into the EcoRI site of pBACe3.6. In total, 157,240 individual BACs have been picked into 384-well plates. Approximately 190 randomly chosen clones have been characterized by Pulsed Field Gel Electrophoresis (PFGE) and have an average insert size of 105 kb, suggesting library coverage representing 5-6 genome equivalents. The frequency of clones without inserts is 4%. The chromosomal location of 51 BACs was studied by FISH; 3 showed more than one signal, indicating a chimerism frequency of roughly 6%. Approximately 50% of the clones in the library contain Simple Repeat Sequences (microsatellites), and 4% of the clones contain centromeric repeats. Insert stability was assessed by restriction digestion of DNA prepared from 20 clones after serial culture for one and three nights. Only one clone showed any evidence of an altered restriction pattern. Clones from 360 x 384-well plates (138,240 colonies) were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones. Both membranes and superpools are available from the RZPD, Berlin (http://www.rzpd.de). PCR 4-D superpools have been prepared from an additional 23,000 clones. The library has been screened for a total of 24 single-copy sequences; positive clones have been obtained in all cases.

Construction and characterisation of a gridded chicken cosmid library with four-fold genomic coverage.

Gridded genomic libraries are crucial for the positional candidate gene approach. For this purpose we constructed a gridded genomic library from a female chicken using the vector sCos 1. About 110,000 cosmid clones were grown and replicated in 384-well plates. An average insert size of 39 kb was calculated from the analysis of 68 randomly selected clones. No chimerism could be observed from 31 in situ hybridisations. One replica of the library (number 125) has been transferred to the Resource Centre/Primary Database (RZPD) of the German Human Genome Project (DHGP). The whole library was gridded onto four nylon filters at high density for efficient identification of cosmid clones by colony hybridisation. Twenty-two probes were used for screening the library and each of them gave at least one positive signal. This result is in good agreement with a four-fold coverage of the genome as estimated from the insert length and number of recombinant clones. This library provides a powerful tool for rapid physical mapping and complex analysis of the chicken genome.

Cell survival following bone-anterior cruciate ligament-bone allograft transplantation: DNA fingerprints, segregation, and collagen morphological analysis of multiple markers in the canine model.

Bone-anterior cruciate ligament-bone allograft transplantation has become recognized as a potential solution to reconstruction of the anterior cruciate ligament (ACL). The purpose of this study was to determine the time-dependent fibrocyte donor cell survival rate after cryopreserved bone-ACL-bone allograft transplantation. Additionally, bony incorporation of the pediculated bone plugs was examined. The ability to successfully transplant allogenous ACL fibrocytes and have them survive has not previously been documented. In this study, DNA fingerprints identified and documented the survival rate of the cellular DNA in transplanted ACL allografts for ACL re-construction in the knee joints of 10 skeletally mature dogs. At 4, 8, 26 and 52 weeks after ACL allograft transplantation, DNA probes, H & E, Giemsa, Goldner, PAS and polarized light staining was done to demonstrate the time-dependent changes in the allografts after transplantation. At 4 weeks host fibrocytes began to grow into the graft; however, histologically the cells could not be distinguished as to host or donor origin. After 4 weeks the DNA pattern reflected only the band pattern of the host. This reveals the early cellular infiltration activity of the host into the ACL allograft, also demonstrated in the light microscopy stainings. The survival rate of transplanted allogenous ACL fibrocytes had not been documented before this study. There is no evidence that ACL allograft cells survive in the intra-articular environment of the host's knee. Within 4 weeks ACL allografts became completely repopulated with host cells. The cells that migrate early into the ACL allografts are probably of synovial origin because they are present before revascularization and collagen reorganization occur. We conclude from this study that viable cells in transplanted ACL allografts did not survive longer than 4 weeks after intra-articular transplantation. Advances in molecular biology may offer new approaches to alter or stimulate fibrocyte population and function in the transplanted ACL allograft used for ACL reconstruction. New methods to maintain the viability of donor cells may be necessary to improve the biomechanical and histological properties of autografts or allografts for ACL reconstruction.

The genetic basis of resistance to Ostertagia circumcincta in lambs.

The relationship between Ostertagia (Teladorsagia) circumcincta and sheep is one of the best understood host-parasite relationships in any species. The key components of resistance have been quantified, the extent of genetic control has been established for lambs, and methods now exist to breed lambs which will be both more resistant to worms and more productive than unselected lambs. A major gene for resistance has been identified within or around the major histocompatibility complex, and this gene appears to be the strongest yet identified for resistance to any parasite species. The most important mechanisms of resistance are local IgA responses which regulate worm fecundity and immediate hypersensitivity responses which regulate worm burdens. IgA responses develop before effective immediate hypersensitivity responses. Good simulation models now exist to predict, for example, outbreaks of disease and the response of sheep to selection. The challenge now is to use our improved understanding of the population biology to develop even better simulation models and to produce expert systems based on these models which can be used by veterinarians and others to determine optimal procedures for individual farms to control disease and reduce sub-clinical economic losses.

Heterogeneity in the recognition of Ostertagia circumcincta antigens by serum antibody from mature, infected sheep.

The recognition of parasite molecules from third-stage and adult Ostertagia circumcincta by serum antibody was studied in a group of matched, mature Scottish Blackface sheep that had been naturally and then deliberately infected. A total of 20 molecules was recognized in somatic extracts from third-stage larvae and 31 molecules in somatic extracts from adult parasites. However, no sheep recognized all immunogenic molecules and no molecule was recognized by all sheep. There was no obvious relationship between recognition of any parasite antigen and polymorphism at class I loci or at the DRBI class II locus of the major histocompatibility complex in these outbred animals. Only 15 molecules from third-stage larvae were present at a frequency suitable for statistical analysis and recognition of three of these 15 molecules was associated with differences in worm burdens. Recognition of two of five molecules from adult parasites was associated with differences in worm length. These results indicate that variation in the recognition of specific, identifiable parasite molecules may be partly responsible for variation among sheep in resistance to O. circumcincta.