R. Fries

Association of a lysine-232/alanine polymorphism in a bovine gene encoding acyl-CoA:diacylglycerol acyltransferase (DGAT1) with variation at a quantitative trait locus for milk fat content

DGAT1 encodes diacylglycerol O-acyltransferase (EC 2.3.1.20), a microsomal enzyme that catalyzes the final step of triglyceride synthesis. It became a functional candidate gene for lactation traits after studies indicated that mice lacking both copies of DGAT1 are completely devoid of milk secretion, most likely because of deficient triglyceride synthesis in the mammary gland. Our mapping studies placed DGAT1 close to the region of a quantitative trait locus (QTL) on bovine chromosome 14 for variation in fat content of milk. Sequencing of DGAT1 from pooled DNA revealed significant frequency shifts at several variable positions between groups of animals with high and low breeding values for milk fat content in different breeds (Holstein–Friesian, Fleckvieh, and Braunvieh). Among the variants was a nonconservative substitution of lysine by alanine (K232A), with the lysine-encoding allele being associated with higher milk fat content. Haplotype analysis indicated the lysine variant to be ancestral. Two animals that were typed heterozygous (Qq) at the QTL based on marker-assisted QTL-genotyping were heterozygous for the K232A substitution, whereas 14 animals that are most likely qq at the QTL were homozygous for the alanine-encoding allele. An independent association study in Fleckvieh animals confirmed the positive effect of the lysine variant on milk fat content. We consider the nonconservative K232A substitution to be directly responsible for the QTL variation, although our genetic studies cannot provide formal proof.

The dynamics of chromosome evolution in birds and mammals.

Comparative mapping, which compares the location of homologous genes in different species, is a powerful tool for studying genome evolution. Comparative maps suggest that rates of chromosomal change in mammals can vary from one to ten rearrangements per million years. On the basis of these rates we would expect 84 to 600 conserved segments in a chicken comparison with human or mouse. Here we build comparative maps between these species and estimate that numbers of conserved segments are in the lower part of this range. We conclude that the organization of the human genome is closer to that of the chicken than the mouse and by adding comparative mapping results from a range of vertebrates, we identify three possible phases of chromosome evolution. The relative stability of genomes such as those of the chicken and human will enable the reconstruction of maps of ancestral vertebrates.

Physically mapped, cosmid-derived microsatellite markers as anchor loci on bovine chromosomes

To identify physical and genetic anchor loci on bovine chromosomes, 13 cosmids, obtained after the screening of partial bovine cosmid libraries with the (CA)n microsatellite motif, were mapped by fluorescence in situ hybridization (FISH). Eleven cosmid probes yielded a specific signal on one of the bovine chromosomes and identified the following loci: D5S2, D5S3, D6S3, D8S1, D11S5, D13S1, D16S5, D17S2, D19S2, D19S3, D21S8. Two cosmids produced centromeric signals on many chromosomes. The microsatellite-containing regions were subcloned and sequenced. The sequence information revealed that the two centromeric cosmids were derived from bovine satellites 1.723 and 1.709, respectively. A cosmid located in the subtelomeric region of Chromosome (Chr) 17 (D17S2) had features of a chromosome-specific satellite. Primers were designed for eight of the nonsatellite cosmids, and seven of these microsatellites were polymorphic with between three and eight alleles on a set of outbred reference families. The polymorphic and chromosomally mapped loci can now be used to physically anchor other bovine polymorphic markers by linkage analysis. The microsatellite primers were also applied to DNA samples of a previously characterized panel of somatic hybrid cell lines, allowing the assignment of seven microsatellite loci to defined syntenic groups. These assignments confirmed earlier mapping results, revealed a probable case of false synteny, and placed two formerly unassigned syntenic groups on specific chromosomes.

Two α(1,2) fucosyltransferase genes on porcine Chromosome 6q11 are closely linked to the blood group inhibitor (S) and Escherichia coli F18 receptor (ECF18R) loci

Abstract. The Escherichia coli F18 receptor locus (ECF18R) has been genetically mapped to the halothane linkage group on porcine Chromosome (Chr) 6. In an attempt to obtain candidate genes for this locus, we isolated 5 cosmids containing the α(1,2)fucosyltransferase genes FUT1, FUT2, and the pseudogene FUT2P from a porcine genomic library. Mapping by fluorescence in situ hybridization placed all these clones in band q11 of porcine Chr 6 (SSC6q11). Sequence analysis of the cosmids resulted in the characterization of an open reading frame (ORF), 1098 bp in length, that is 82.3% identical to the human FUT1 sequence; a second ORF, 1023 bp in length, 85% identical to the human FUT2 sequence; and a third FUT-like sequence thought to be a pseudogene. The FUT1 and FUT2 loci therefore seem to be the porcine equivalents of the human blood group H and Secretor loci. Direct sequencing of the two ORFs in swine being either susceptible or resistant to adhesion and colonization by F18 fimbriated Escherichia coli (ECF18) revealed two polymorphisms at bp 307 (M307) and bp 857 (M857) of the FUT1 ORF. Analysis of these mutations in 34 Swiss Landrace families with 221 progeny showed close linkage with the locus controlling resistance and susceptibility to E. coli F18 adhesion and colonization in the small intestine (ECF18R), and with the locus of the blood group inhibitor S. A high linkage disequilibrium of M307–ECF18R in Large White pigs makes the M307 mutation a good marker for marker-assisted selection of E. coli F18 adhesion-resistant animals in this breed. Whether the FUT1 or possibly the FUT2 gene products are involved in the synthesis of carbohydrate structures responsible for bacterial adhesion remains to be determined.

A major genetic component of BSE susceptibility

BackgroundCoding variants of the prion protein gene (PRNP) have been shown to be major determinants for the susceptibility to transmitted prion diseases in humans, mice and sheep. However, to date, the effects of polymorphisms in the coding and regulatory regions of bovine PRNP on bovine spongiform encephalopathy (BSE) susceptibility have been considered marginal or non-existent. Here we analysed two insertion/deletion (indel) polymorphisms in the regulatory region of bovine PRNP in BSE affected animals and controls of four independent cattle populations from UK and Germany.ResultsIn the present report, we show that two previously reported 23- and 12-bp insertion/deletion (indel) polymorphisms in the regulatory region of bovine PRNP are strongly associated with BSE incidence in cattle. Genotyping of BSE-affected and control animals of UK Holstein, German Holstein, German Brown and German Fleckvieh breeds revealed a significant overrepresentation of the deletion alleles at both polymorphic sites in diseased animals (P = 2.01 × 10-3 and P = 8.66 × 10-5, respectively). The main effect on susceptibility is associated with the 12-bp indel polymorphism. Compared with non-carriers, heterozygous and homozygous carriers of the 12-bp deletion allele possess relatively higher risks of having BSE, ranging from 1.32 to 4.01 and 1.74 to 3.65 in the different breeds. These values correspond to population attributable risks ranging from 35% to 53%.ConclusionOur results demonstrate a substantial genetic PRNP associated component for BSE susceptibility in cattle. Although the BSE risk conferred by the deletion allele of the 12-bp indel in the regulatory region of PRNP is substantial, the main risk factor for BSE in cattle is environmental, i.e. exposure to feedstuffs contaminated with the infectious agent.

Variation in neighbouring genes of the dopaminergic and serotonergic systems affects feather pecking behaviour of laying hens

Feather pecking is a behavioural disorder of laying hens and has serious animal welfare and economic implications. One of the several aetiological hypotheses proposes that the disorder results from redirected exploratory behaviour. Variation in the gene encoding the dopamine D4 receptor (DRD4) has been shown to be associated with exploratory behaviour in several species, including in a passerine bird species. We therefore considered DRD4 as a candidate gene for feather pecking. We have annotated DRD4 in the chicken genome and have re-sequenced it in 140 animals belonging to: experimental layer lines divergently selected for high and low propensity to feather pecking; the unselected founder population; and two commercial lines with low and high propensity to feather pecking. We have identified two sub-haplotypes of DRD4 that are highly significantly associated with feather pecking behaviour in the experimental (P = 7.30 x 10(-7)) as well as in the commercial lines (P = 2.78 x 10(-6)). Linkage disequilibrium (LD) extends into a neighbouring gene encoding deformed epidermal autoregulatory factor 1 (DEAF1). The product of DEAF1 regulates the transcription of the gene encoding the serotonin (5-hydroxytryptamine) 1A receptor. Thus, DEAF1 represents another candidate gene for feather pecking. Re-sequencing of five animals homozygous for the low-pecking sub-haplotype and of six animals homozygous for the high-pecking sub-haplotype delineated an LD block of 14 833 bases spanning the two genes. None of the variants in the LD block is obviously functional. However, the haplotype information will be useful to select against the propensity to feather pecking in chicken and to elucidate the functional implications of the variants.

Combined Q-banding and fluorescence in situ hybridization for the identification of bovine chromosomes 1 to 7.

Eleven probes were assigned to bovine chromosomes 1 to 7 by fluorescence in situ hybridization (FISH). The identification of chromosomes was based on QFQ-banding prior to in situ hybridization and comparison with the Reading Conference (1976) and ISCNDA (1989) standards. The probes used for FISH can now be utilized as identification and discrimination features for bovine chromosomes 1 to 7 and particularly for chromosomes 4 and 6, which are difficult to distinguish. Comparison of our mapping data with previous assignments and of the standard chromosome banding patterns prompt us to propose a change in the ISCNDA nomenclature: ISCNDA chromosome 4 should be named chromosome 6 and vice versa. Chromosome 4 is marked by the ribosomal RNA cluster RNR3, and chromosome 6 is characterized by the casein gene cluster and an anonymous satellite (D6Z1).

Cosmid-derived markers anchoring the bovine genetic map to the physical map.

The mapping strategy for the bovine genome described in this paper uses large insert clones as a tool for physical mapping and as a source of highly polymorphic microsatellites for genetic typing, and was one objective of the BovMap Project funded by the European Union (UE). Eight-three cosmid and phage clones were characterized and used to physically anchor the linkage groups defining all the bovine autosomes and the X Chromosome (Chr). By combining physical and genetic mapping, clones described in this paper have led to the identification of the linkage groups corresponding to Chr 9, 12, 16, and 25. In addition, anchored loci from this study were used to orient the linkage groups corresponding to Chr 3, 7, 8, 9, 13, 16, 18, 19, and 28 as identified in previously published maps. Comparison of the estimated size of the physical and linkage maps suggests that the genetic length of the bovine genome may be around 4000 cM.

The bovine lactoferrin gene (LTF) maps to Chromosome 22 and syntenic group U12

Five overlapping lambda EMBL-clones, containing the complete bovine lactoferrin gene (LTF), have been used to map this gene by fluorescence in situ hybridization to bovine Chromosome (Chr) band 22q24. Primers derived from promoter and exon I sequences were applied in polymerase chain reactions (PCRs) to DNA samples of a previously characterized panel of somatic cell hybrid lines, allowing the assignment of the bovine lactoferrin locus to syntenic group U12. These results permit the assignment of syntenic group U12 to bovine Chr 22.

Physical and linkage mapping of the bovine genome with cosmids

We have initiated a mapping strategy using cosmid clones to chromosomally anchor a high-resolution bovine genetic linkage map. Ten cosmids containing microsatellites were assigned to bovine chromosomes by fluorescence in situ suppression hybridization (FISH). Four cosmid clones, three of which contain an informative microsatellite, were assigned to autosomes 5, 13, 24, and 28. The assignment to autosome 13 anchors bovine syntenic group U11. Two additional cosmid clones, each containing informative microsatellites, are assigned to autosomes 9 and 29, auchoring bovine linkage groups U2 and U8, respectively. Four cosmid clones, three of which contain informative microsatellites, also provide the first assignment to autosome 25, anchoring bovine syntenic group U7 and orienting the corresponding linkage group relative to the centromere.