A chromosome-wide QTL study on BTA29 affecting temperament traits in German Angus beef cattle and mapping of DRD4.

The behaviour of beef cattle is important for the safety and welfare of stockmen and animals. Ten microsatellites spanning BTA29 and, in addition, the candidate gene, dopamine receptor D4 gene, were analysed in 545 German Angus calves of six sires and included in a quantitative trait locus (QTL) study on the basis of three different behaviour tests. A putative QTL for the score while entering the scale (ScE) was detected at BMS764. The DRD4 fragment was mapped in the distal region of BTA29 15.3 cM distal of ILSTS081. The results clearly indicate that BTA29 with a putative QTL in the proximal part and the candidate gene, DRD4, in the distal part plays an important role in the regulation of temperament. During the study one of the sires was detected to be a blood chimera.

Genetic variation in the kappa-casein gene (CSN3) of Chinese yak (Bos grunniens) and phylogenetic analysis of CSN3 sequences in the genus Bos.

Variants of kappa-casein (CSN3) have been extensively studied in cattle and 13 alleles have been identified at the protein and DNA levels to date. Evolution of some of these alleles and a possible common ancestor remain unclear. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of CSN3 exon IV in domesticated yak revealed a 2-allele polymorphism showing migration patterns different from known cattle variants. The PCR products of both yak CSN3 SSCP alleles were sequenced. All yak had nucleotide sequences coding for Thr in AA position 136 (identical to bovine CSN3*A) and Ala in position 148 (identical to bovine CSN3*B). The sequencing results were confirmed by PCR-RFLP analysis using HindIII and TaqI. A 12-bp insertion in the coding region, representing a repeated nucleotide and AA motif, was found in 1 yak allele. The duplication corresponds to the codons for AA 147 to 150 (Glu-Ala-Ser-Pro) or 148 to 151 (Ala-Ser-Pro-Glu), which are repeated identically. In 21 yak samples genotyped by PCR-SSCP analysis, frequencies for the insertion variant and the short variant were about 68 and 32%, respectively. The loss of the insertion may have led to the ancestral CSN3 allele from which all currently known variants of CSN3 in the genus Bos evolved. This is the first report of polymorphisms in the yak CSN3 gene and may be helpful for future studies on genetic variation within and between yak populations or on associated traits.

Molecular characterization of bovine CSN1S2*B and extensive distribution of zebu-specific milk protein alleles in European cattle.

The B allele of the bovine alpha (S2)-casein gene (CSN1S2) was characterized at the molecular level and the distribution of zebu-specific milk protein alleles was determined in 26 cattle breeds originating from 3 continents. The CSN1S2*B allele is characterized by a C --> T transition affecting nucleotide 17 of exon 3, which leads to a change in the eighth amino acid of the mature protein, from Ser to Phe (i.e., TCC --> TTC). DNA-based methods were developed to identify carriers of CSN1S2*B and the other alleles (CSN1S2*A, C, and D) at the same locus. CSN1S2*B and other zebu-specific milk protein alleles and casein haplotypes are widely distributed in European cattle breeds, particularly those of southeastern origin. Alleles CSN1S2*B and CSN3*H are important in searching for zebu imprints in European cattle breeds. Diversity estimates at the milk protein loci were highest in the zebus followed by southeastern European taurines. Anatolian Black had the highest number of zebu alleles among European taurines. Common, group, and intergroup haplotypes occurred in the breeds and demonstrated relationships that concurred with developmental histories, genetic makeup, and, in particular, exposed the extent of zebu influence on southeastern European cattle.

Joint analysis of the influence of CYP11B1 and DGAT1 genetic variation on milk production, somatic cell score, conformation, reproduction, and productive lifespan in German Holstein cattle.

Recent publications indicate genetic variation in milk production traits on proximal BTA14, which cannot be explained solely with genetic variation in the DGAT1 gene. To elucidate these QTL effects, animals from a German Holstein granddaughter design (18 families, 1,291 sons) were genotyped for CYP11B1 (V30A) and DGAT1 (K232A) polymorphisms. Frequencies of alleles of maternal descent were estimated for CYP11B1(V) (0.776) and DGAT1(K) (0.549). Allele substitution effects (alpha/2) were first calculated for both alleles in separate models and then in a joint model. From the joint analysis, CYP11B1(V) effects on fat content (+0.04%) and protein content (+0.01%) were positive. Effects on milk yield (-82 kg), fat yield (-0.5 kg), and protein yield (-1.9 kg) were negative. Compared with the individual analysis, DGAT1(K) effects on fat content (+0.28%), protein content (+0.06%), and milk yield (-258 kg) were reduced; fat yield (+10.8 kg) was enhanced; and protein yield (-3.8 kg) was reduced. In the joint analysis, allele substitution effects of CYP11B1(V) and DGAT1(K) together explained more of the variation in milk production traits than DGAT1(K) alone. Further significant effects were found for CYP11B1(V) and DGAT1(K) among 6 reproduction traits and 14 conformational traits. These observations indicate a possible negative influence of DGAT1(K) on maternal nonreturn rate, and thus, on length of productive life.

Characterization of the DGAT1 mutations and the CSN1S1 promoter in the German Angeln dairy cattle population.

The identification of quantitative trait loci (QTL) and genes with influence on milk production traits has been the objective of various mapping studies in the last decade. In the centromeric region of Bos taurus autosome (BTA) 14, the acyl-CoA:diacylglycerol acyltransferase1 gene (DGAT1) has been identified as the most likely causative gene underlying a QTL for milk fat yield and content. Recently, a second polymorphism in the promoter of DGAT1 emerged as an additional source of variation. In this study, the frequencies and the effects of alleles at the DGAT1 K232A and at the DGAT1 promoter variable number of tandem repeat (VNTR) locus on BTA14, and of alleles at the CSN1S1 (alpha(S1)-casein-encoding gene) promoter on BTA6 in the German Angeln dairy cattle population were investigated. Analyzed traits were milk, fat, protein, lactose, and milk energy yield, fat, protein, lactose, and milk energy content and somatic cell score. The lysine variant of the DGAT1 K232A mutation showed significant effects for most of the milk production traits. A specific allele of the DGAT1 promoter VNTR showed significant effects on the traits lactose yield and content, milk energy content, and SCS compared with the other alleles. Additionally, a regulation mechanism between the DGAT1 K232A mutation and the DGAT1 promoter VNTR was found for fat yield and content, which could be caused by an upper physiological bound for the effects of the DGAT1 gene. At the CSN1S1 promoter, 2 of 4 alleles showed significant allele substitution effects on the milk yield traits.

Caprine kappa-casein (CSN3) polymorphism: new developments in molecular knowledge.

A high degree of polymorphism was recently found at the kappa-casein (CSN3) locus in the domesticated goat (Capra hircus). In the present study, 2 new patterns previously identified by PCR-single-strand conformation polymorphism analysis (SSCP) were characterized. The allele provisionally named "X" (GenBank Accession no. AY350425) differs from CSN3*C (AF485341) by a (silent) A-->G substitution at position 509 of the goat CSN3 reference sequence (X60763). As this newly identified sequence changes the amino acid sequence, and the already known CSN3*C allele (AF485341) has an additional silent mutation, we proposed a change in nomenclature to reflect these changes, indicating the silent mutation with the prime symbol (i.e.,'). The CSN3*M allele (provisionally named "Y") results in a new protein variant, differing by 2 nonsynonymous mutations from the CSN3*F allele. The new variant is characterized by a G-->A transition at nucleotide position 384, resulting in the amino acid exchange Asp90-->Asn90, and a C-->T transition at position 550, resulting in a Val145-->Ala145 substitution. Thus, the number of alleles identified in the domesticated goat has increased to 16, of which 13 are protein variants and 3 are silent mutations, involving a total of 15 polymorphic sites in CSN3 exon 4. Data on the distribution of the main alleles in 7 goat breeds of Europe, West Africa, and the Near East show differences in the occurrence and frequency of the alleles between breeds and geographic origin with the highest number of alleles found in goat breeds from the Near East.

Polymorphism of the bovine CSN1S1 promoter: linkage mapping, intragenic haplotypes, and effects on milk production traits.

The bovine CSN1S1 5' flanking region (CSN1S1-5') was screened for polymorphisms in different cattle breeds. Single-strand conformation polymorphisms (SSCP) and sequence analyses revealed four alleles (1-4), two of them being new allelic forms (3 and 4). Sequences were deposited in GenBank with accession numbers AF549499-502. In alleles 1 and 4, potential transcription factor binding sites are altered by the mutations. Using SSCP analysis, all four alleles were identified in German Holsteins. Six intragenic haplo-types comprising CSN1S1-5' (alleles 1, 2, 3, 4) and exon 17 (CSN1S1*B and C) genotypes were found. Linkage mapping using half-sib families from the German QTL project positioned CSN1S1 between the markers FBN14 and CSN3, with 5.6 cM distance between CSN1S1 and CSN3. Variance analysis, using family and CSN1S1 promoter genotypes as fixed effects, of breeding values and deregressed proofs for milk production traits (milk, fat, and protein yield and also fat and protein percentage) revealed significant effects on protein percentage when all families and genotypes were considered. Contrast calculations assigned a highly significant effect to genotype 24, which was associated with highest LS-means for protein percentage breeding values. As CSN1S1 is one of the main caseins in milk, this could be an effect of mutations in regulatory elements in the promoter region. An effect on milk yield breeding values was indicated for genotype 12, but is probably caused by a linked locus.

Detection of casein fragments in an invertebrate and in a vertebrate using in situ hybridization.

Using a non-radioactive in situ hybridization procedure it has been demonstrated that both invertebrates such as the mollusc Mytilus galloprovincialis and lower vertebrates such as the fish Cyprinus carpio express bovine alphaS1- and kappa-casein homologous mRNAs. In particular, positive results were found in molluscan immunocytes, and in cells located in different fish tissues: intestine, endocrine pancreas and kidney. These findings suggest that the casein genes are highly conserved throughout evolution.

SSCP analysis at the bovine CSN3 locus discriminates six alleles corresponding to known protein variants (A, B, C, E, F, G) and three new DNA polymorphisms (H, I, A1).

A high resolution SSCP protocol was developed for simultaneous discrimination of the known CSN3 alleles A, B, C, E, F and G. Furthermore, three new DNA polymorphisms were identified in different Bos taurus and Bos indicus breeds or crosses. Mendelian segregation was shown for two of these polymorphisms (named CSN3*H and 1), and the third (named CSN3*A1) was found in unrelated animals, thus indicating the presence of three additional alleles at the bovine CSN3 locus. DNA sequencing revealed single mutations that led to a Thr/Ile substitution in amino acid position 135 for CSN3*H and to a Ser/Ala substitution in position 104 of the deduced amino acid sequence of CSN3*1 (GenBank accession numbers AF105260 and AF121023) compared to CSN3*A. In CSN3*A1, a silent mutation in the third codon position of Pro150 was found (GenBank accession number AF092513).

Biochemical characterization of bovine alpha s1-casein F and genotyping with sequence-specific primers.

Bovine alpha s1-casein F (alpha s1-CN F) was found in a genetic resource of Deutsches Schwarzbuntes Niederungsrind cows at a frequency of 0.009. Biochemical characterization of this new variant was obtained by automated sequencing of reversed-phase HPLC-separated tryptic peptides of alpha s1-CN F and alpha s1-CN B. alpha s1-CN F was found to be a subtype of alpha s1-CN B with a single amino acid substitution (SerP/Leu) in position 66. DNA sequencing revealed a C/T transition in position 8418 of the gene. Sequence-specific primers were designed to perform an allele-specific polymerase chain reaction for detection of alpha s1 CnF. Typing of artificial insemination sperm samples included in the genetic resource sperm pool identified one sire heterozygous for alpha s1 CnF.

Molecular genetic characterization of new bovine kappa-casein alleles CSN3F and CSN3G and genotyping by PCR-RFLP.

In order to characterize the two new kappa-casein variants F and G (CSN3F and CSN3G) recently detected in Ayrshire and Pinzgauer cattle, exon IV of CSN3 from heterozygous animals was amplified by polymerase chain reaction (PCR), cloned and sequenced. The sequencing data revealed single point mutations at nucleotide positions 10530 (G-->A) for CSN3F and 10790 (C-->T) for CSN3G, corresponding to amino acid exchanges in positions 10 (Arg-->His) and 97 (Arg-->Cys) respectively. These mutations alter recognition sites for the restriction enzymes HhaI and MaeII, which were subsequently used to confirm these polymorphisms in cattle carrying CSN3F or CSN3G. A PCR-restriction fragment length polymorphism (RFLP) genotyping procedure for all currently known CSN3 alleles (CSN3A, CSN3B, CSN3C, CSN3E, CSN3F, CSN3G) was developed.