The role of the bovine growth hormone receptor and prolactin receptor genes in milk, fat and protein production in Finnish Ayrshire dairy cattle

We herein report new evidence that the QTL effect on chromosome 20 in Finnish Ayrshire can be explained by variation in two distinct genes, growth hormone receptor (GHR) and prolactin receptor (PRLR). In a previous study in Holstein-Friesian dairy cattle an F279Y polymorphism in the transmembrane domain of GHR was found to be associated with an effect on milk yield and composition. The result of our multimarker regression analysis suggests that in Finnish Ayrshire two QTL segregate on the chromosomal region including GHR and PRLR. By sequencing the coding sequences of GHR and PRLR and the sequence of three GHR promoters from the pooled samples of individuals of known QTL genotype, we identified two substitutions that were associated with milk production traits: the previously reported F-to-Y substitution in the transmembrane domain of GHR and an S-to-N substitution in the signal peptide of PRLR. The results provide strong evidence that the effect of PRLR S18N polymorphism is distinct from the GHR F279Y effect. In particular, the GHR F279Y has the highest influence on protein percentage and fat percentage while PRLR S18N markedly influences protein and fat yield. Furthermore, an interaction between the two loci is suggested.     

A QTL affecting milk yield and composition maps to bovine chromosome 20: a confirmation

As part of a whole genome scan undertaken to detect quantitative trait loci (QTL) affecting milk yield and composition, we have genotyped a granddaughter design comprising 1152 sons for six microsatellite markers spanning bovine chromosome 20. An analysis performed across families provided strong evidence (experiment-wise P -values < 0·01) for the presence of a QTL affecting primarily protein percentage towards the telomeric end of the chromosome. A founder sire, shown in a previous study to segregate for a similar QTL in the corresponding chromosome region, was characterized by 29 and 57 sons and maternal grandsons, respectively, in the present design. Sorting corresponding sons and grandsons by paternal or grandpaternal allele provided significant evidence for the segregation of a QTL on chromosome 20. Altogether these results confirm the location of a QTL affecting milk production on bovine chromosome 20.     

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.     

The identification of QTL that affect the fatty acid composition of milk on sheep chromosome 11

In this work, we analysed 11 genetic markers localized on OAR11 in a commercial population of Spanish Churra sheep to detect QTL that underlie milk fatty acid (FA) composition traits. Following a daughter design, we analysed 799 ewes distributed in 15 half‐sib families. Eight microsatellite markers and three novel SNPs identified in two genes related to fatty acid metabolism, acetyl‐CoA carboxylase α (ACACA) and fatty acid synthase (FASN), were genotyped in the whole population under study. The phenotypic traits considered in the study included 22 measurements related to the FA composition of the milk and three other milk production traits (milk protein percentage, milk fat percentage and milk yield). Across‐family regression analysis revealed four significant QTL at the 5% chromosome‐wise level influencing contents of capric acid (C10:0), lauric acid (C12:0), linoleic conjugated acid (CLA) and polyunsaturated fatty acids (PUFA) respectively. The peaks of the QTL affecting C10:0 and PUFA contents in milk map close to the FASN gene, which has been evaluated as a putative positional candidate for these QTL. The QTL influencing C12:0 content reaches its maximum significance at 58 cM, close to the gene coding for the glucose‐dependent insulinotropic polypeptide. We were not able to find any candidate genes related to fat metabolism at the QTL influencing CLA content, which is located at the proximal end of the chromosome. Further research efforts will be needed to confirm and refine the QTL locations reported here.     

Effects of DGAT1 and GHR on milk yield and milk composition in the Chinese dairy population

Previous studies have demonstrated that the p.Lys232Ala substitution in the acylCoA: diacylglycerol acyltransferase ( DGAT1 ) gene and the p.Phe279Tyr mutation in the growth hormone receptor ( GHR ) gene are the causative quantitative trait loci underlying milk yield and composition on BTA14 and BTA20 respectively. To examine their applications in the genetic improvement of Chinese dairy cattle productivity, we herein investigated the effects of the DGAT1 p.Lys232Ala and GHR p.Phe279Tyr mutations on milk, fat and protein yield, as well as fat and protein percentage in the milk of 1222 Holstein cows. Genotyping was performed using PCR-RFLP for DGAT1 or primer-introduced restriction analysis (PCR-PIRA) for GHR . With a mixed animal model, the significant associations of the DGAT1 p.Lys232Ala substitution with 305-day milk, fat and protein yield were identified ( P  =   0.0001). The DGAT1 allele that encode lysine at position 232 was associated with increased 305-day milk fat yield, but with decreased 305-day milk and protein yield, whereas the GHR p.Phe279Tyr mutation was found to be significantly associated with protein percentage ( P  =   0.0014). The allele substitution effect of p.279Phe by p.279Tyr may lead to a significant increase in protein percentage. Our findings indicate that DGAT1 p.232Ala and GHR p.279Phe could be used to increase milk yield and protein yield of Chinese Holstein cows.     

A mammary gland EST showing linkage disequilibrium to a milk production QTL on bovine Chromosome 14

As part of a genome scan, ESTs derived from mammary gland tissue of a lactating cow were used as candidate genes for quantitative trait loci (QTL), affecting milk production traits. Resource families were genotyped with 247 microsatellite markers and 4 polymorphic ESTs. It was shown by linkage analysis that one of these ESTs, KIEL_E8, mapped to the centromeric region of bovine Chromosome (Chr) 14. Regression analysis revealed the presence of a QTL, with significant effect on milk production, in this chromosome region, and analysis of variance showed no significant interaction of marker genotype and family. The estimated significant differences between homozygous marker genotypes were 140 kg milk, −5.02 kg fat yield, and 2.58 kg protein yield for the first 100 days of lactation. Thus, there was strong evidence for a complete or nearly complete linkage disequilibrium between KIEL_E8 and the QTL. To identify the biological function of KIEL_E8, we extended the sequence for 869 bp by 5′-RACE. A 560-bp fragment of this shows a 90.9% similarity to a gene encoding a cysteine- and histidine-rich cytoplasmic protein in mouse. Although such a protein may have a regulatory function for lactation and a linkage disequilibrium between the EST marker and the QTL has been observed, it remains to be elucidated whether they are identical or not. Nevertheless, KIEL_E8 will be an efficient marker to perform marker-assisted selection in the Holstein-Friesian population. Received 20 October 2000 / Accepted: 11 April 2001     

A Triad of Highly Divergent Polymeric Immunoglobulin Receptor (PIGR) Haplotypes with Major Effect on IgA Concentration in Bovine Milk

The aim of this study was to determine a genetic basis for IgA concentration in milk of Bos taurus. We used a Holstein-Friesian x Jersey F2 crossbred pedigree to undertake a genome-wide search for QTL influencing IgA concentration and yield in colostrum and milk. We identified a single genome-wide significant QTL on chromosome 16, maximising at 4.8 Mbp. The polymeric immunoglobulin receptor gene (PIGR) was within the confidence interval of the QTL. In addition, mRNA expression analysis revealed a liver PIGR expression QTL mapping to the same locus as the IgA quantitative trait locus. Sequencing and subsequent genotyping of the PIGR gene revealed three divergent haplotypes that explained the variance of both the IgA QTL and the PIGR expression QTL. Genetic selection based on these markers will facilitate the production of bovine herds producing milk with higher concentrations of IgA.     

Molecular anatomy of the cytoplasmic domain of bovine growth hormone receptor, a quantitative trait locus

Quantitative trait loci (QTL) studies have indicated growth hormone receptor (GHR) as a candidate gene affecting cattle milk yield and composition. In order to characterize genetic variation at GHR in cattle, we studied European and East African breeds with different histories of selection, and Bos grunniens, Ovis aries, Sus scrofa, Bison bison and Rangifer tarandus as references. We sequenced most of the cytoplasmic domain (900 bp of exon 10), 89 bp of exon 8, including the putative causative mutation for the QTL effect, and 390 bp of intron 8 for comparison. In the cytoplasmic domain, seven synonymous and seven non-synonymous single nucleotide polymorphisms (SNP) were identified in cattle. Three non-synonymous SNPs were found in sheep and one synonymous SNP in yak, while other studied species were monomorphic. Three major haplotypes were observed, one unique to African breeds, one unique to European breeds and one shared. Bison and yak haplotypes are derivatives of the European haplotype lineage. Most of the exon 10 non-synonymous cattle SNPs appear at phylogenetically highly conserved sites. The polymorphisms in exon 10 cluster around a ruminant-specific tyrosine residue, suggesting that this site may act as an additional signalling domain of GHR in ruminants. Alternative explanations for the persistent polymorphism include balancing selection, hitch-hiking, pleiotropic or sexually antagonistic fitness effects or relaxed functional constraints.     

A 12 kb multi-allelic copy number variation encompassing a GC gene enhancer is associated with mastitis resistance in dairy cattle

Clinical mastitis (CM) is an inflammatory disease occurring in the mammary glands of lactating cows. CM is under genetic control, and a prominent CM resistance QTL located on chromosome 6 was reported in various dairy cattle breeds. Nevertheless, the biological mechanism underpinning this QTL has been lacking. Herein, we mapped, fine-mapped, and discovered the putative causal variant underlying this CM resistance QTL in the Dutch dairy cattle population. We identified a ~12 kb multi-allelic copy number variant (CNV), that is in perfect linkage disequilibrium with a lead SNP, as a promising candidate variant. By implementing a fine-mapping and through expression QTL mapping, we showed that the group-specific component gene (GC), a gene encoding a vitamin D binding protein, is an excellent candidate causal gene for the QTL. The multiplicated alleles are associated with increased GC expression and low CM resistance. Ample evidence from functional genomics data supports the presence of an enhancer within this CNV, which would exert cis-regulatory effect on GC. We observed that strong positive selection swept the region near the CNV, and haplotypes associated with the multiplicated allele were strongly selected for. Moreover, the multiplicated allele showed pleiotropic effects for increased milk yield and reduced fertility, hinting that a shared underlying biology for these effects may revolve around the vitamin D pathway. These findings together suggest a putative causal variant of a CM resistance QTL, where a cis-regulatory element located within a CNV can alter gene expression and affect multiple economically important traits.     

A QTL with major effect on milk yield and composition maps to bovine Chromosome 14

A whole genome scan was undertaken in a granddaughter design comprising 1158 progeny-tested bulls in order to map QTL influencing milk yield and composition. In this paper we report the identification of a locus on the centromeric end of bovine Chromosome (Chr) 14, with major effect on fat and protein percentage as well as milk yield. The genuine nature of this QTL was verified using the grand²-daughter design, that is, by tracing the segregating QTL alleles from heterozygous grandsires to their maternal grandsons and confirming the predicted QTL allele substitution effect. Received: 30 December 1997 / Accepted: 21 February 1998     

A Point Mutation in Suppressor of Cytokine Signalling 2 (Socs2) Increases the Susceptibility to Inflammation of the Mammary Gland while Associated with Higher Body Weight and Size and Higher Milk Production in a Sheep Model

Mastitis is an infectious disease mainly caused by bacteria invading the mammary gland. Genetic control of susceptibility to mastitis has been widely evidenced in dairy ruminants, but the genetic basis and underlying mechanisms are still largely unknown. We describe the discovery, fine mapping and functional characterization of a genetic variant associated with elevated milk leukocytes count, or SCC, as a proxy for mastitis. After implementing genome-wide association studies, we identified a major QTL associated with SCC on ovine chromosome 3. Fine mapping of the region, using full sequencing with 12X coverage in three animals, provided one strong candidate SNP that mapped to the coding sequence of a highly conserved gene, suppressor of cytokine signalling 2 (Socs2). The frequency of the SNP associated with increased SCC was 21.7% and the Socs2 genotype explained 12% of the variance of the trait. The point mutation induces the p.R96C substitution in the SH2 functional domain of SOCS2 i.e. the binding site of the protein to various ligands, as well-established for the growth hormone receptor GHR. Using surface plasmon resonance we showed that the p.R96C point mutation completely abrogates SOCS2 binding affinity for the phosphopeptide of GHR. Additionally, the size, weight and milk production in p.R96C homozygote sheep, were significantly increased by 24%, 18%, and 4.4%, respectively, when compared to wild type sheep, supporting the view that the point mutation causes a loss of SOCS2 functional activity. Altogether these results provide strong evidence for a causal mutation controlling SCC in sheep and highlight the major role of SOCS2 as a tradeoff between the host’s inflammatory response to mammary infections, and body growth and milk production, which are all mediated by the JAK/STAT signaling pathway.     

Quantitative trait loci mapping in dairy cattle: review and meta-analysis

From an extensive review of public domain information on dairy cattle quantitative trait loci (QTL), we have prepared a draft online QTL map for dairy production traits. Most publications (45 out of 55 reviewed) reported QTL for the major milk production traits (milk, fat and protein yield, and fat and protein concentration (%)) and somatic cell score. Relatively few QTL studies have been reported for more complex traits such as mastitis, fertility and health. The collated QTL map shows some chromosomal regions with a high density of QTL, as well as a substantial number of QTL at single chromosomal locations. To extract the most information from these published records, a meta-analysis was conducted to obtain consensus on QTL location and allelic substitution effect of these QTL. This required modification and development of statistical methodologies. The meta-analysis indicated a number of consensus regions, the most striking being two distinct regions affecting milk yield on chromosome 6 at 49 cM and 87 cM explaining 4.2 and 3.6 percent of the genetic variance of milk yield, respectively. The first of these regions (near marker BM143) affects five separate milk production traits (protein yield, protein percent, fat yield, fat percent, as well as milk yield).     

Confirmation and refinement of a QTL on BTA5 affecting milk production traits in the Fleckvieh dual purpose cattle breed

We analysed a QTL affecting milk yield (MY), milk protein yield (PY) and milk fat yield (FY) in the dual purpose cattle breed Fleckvieh on BTA5. Twenty-six microsatellite markers covering 135 cM were selected to analyse nine half-sib families containing 605 sons in a granddaughter design. We thereby assigned two new markers to the public linkage map using the CRI-MAP program. Phenotypic records were daughter yield deviations (DYD) originating from the routinely performed genetic evaluations of breeding animals. To determine the position of the QTL, three different approaches were applied: interval mapping (IM), linkage analysis by variance component analysis (LAVC), and combined linkage disequilibrium (LD) and linkage (LDL) analysis. All three methods mapped the QTL in the same marker interval ( BM2830-ETH152 ) with the greatest test-statistic value at 118, 119.33 and 119.33 cM respectively. The positive QTL allele simultaneously increases DYD in the first lactation by 272 kg milk, 7.1 kg milk protein and 7.0 kg milk fat. Although the mapping accuracy and the significance of a QTL effect increased from IM over LAVC to LDL, the confidence interval was large (13, 20 and 24 cM for FY, MY and PY respectively) for the positional cloning of the causal gene. The estimated averages of pair wise marker LD with a distance <5 cM were low (0.107) and reflect the large effective population size of the Fleckvieh subpopulation analysed. This low level of LD suggests a need for increase in marker density in following fine mapping steps.     

Detection of QTL for milk production on Chromosomes 1 and 6 of Holstein cattle

Seventy to 75 sons of each of six Holstein sires were assayed for genotypes at a number of microsatellite loci spanning Chromosomes (Chrs) 1 and 6. The number of informative loci varied from three to eight on each chromosome in different sire families. Linkage order and map distance for microsatellite loci were estimated using CRI-MAP. Estimates of QTL effect and location were made by using a least squares interval mapping approach based on daughter yield deviations of sons for 305-d milk, fat, protein yield, and fat and protein percentage. Thresholds for statistical significance of QTL effects were determined from interval mapping of 10,000 random permutations of the data across the bull sire families and within each sire family separately. Across-sire analyses indicated a significant QTL for fat and protein yield, and fat percentage on Chr 1, and QTL effects on milk yield and protein percentage that might represent one or two QTL on Chr 6. Analyses within each sire family indicated significant QTL effects in five sire families, with one sire possibly being heterozygous for two QTLs. Statistically significant estimates of QTL effects on breeding value ranged from 340 to 640 kg of milk, from 15.6 to 28.4 kg of fat, and 14.4 to 17.6 kg of protein. Received: 19 November 1999 / Accepted: 31 August 2000     

Characterization and RH mapping of bovine microsatellites generated from a microdissected BTA20-specific DNA library

Bovine chromosome 20 (BTA20) is associated with several quantitative trait loci (QTL) for meat tenderness, birth weight, milk yield and composition. Fine mapping of these QTL requires the development of additional informative markers to increase the resolution of the BTA20 genetic and physical maps. A BTA20-specific library was constructed by means of microdissection and microcloning, and screened for dinucleotide repeats with (CA)16 and (GT)16 oligos. A total of 60 new microsatellites (MS) were developed and characterized for polymorphism using the U.S. Department of Agriculture (USDA)/Meat Animal Research Center (MARC) bovine reference family, of which 53 markers were informative in this family. The number of alleles for these loci varied from 1 to 14, with an average of 6.5. Thirty-three of these MSs, together with 105 markers previously mapped to BTA20, were scored on a 7000-rad cattle-hamster whole-genome radiation hybrid panel (SUNbRH), resulting in a high-resolution RH7000 rad map for BTA20.     

A search for quantitative trait loci for milk production traits on chromosome 6 in Finnish Ayrshire cattle

Cattle chromosome 6 was scanned with 11 markers, ten microsatellites and the casein haplotype, to identify quantitative trait loci (QTLs) affecting the following milk production traits: milk yield, fat percentage, fat yield, protein percentage and protein yield. Twelve Finnish Ayrshire half-sib families with a total of 480 sons were genotyped and used in a grand-daughter design. Interval mapping was performed with a multiple-marker regression approach with a one-QTL and a two-QTL model, and the significance threshold values were determined empirically using a permutation test. Across-family analysis with the one-QTL model revealed an effect on protein percentage (P < 0.05) and on milk yield (P < 0.05). The analysis with the two-QTL model identified significant effects (P < 0.05) on protein percentage, milk yield, and fat yield. Comparing these two cases, the results suggest the existence of two QTLs on chromosome 6 with an effect on milk production traits. One of the QTLs was located around the casein genes. As the other QTL was similar in location and effect to a QTL found previously in Holstein-Friesians, an identity-by-descent approach could be applied to fine map this region.     

A non-synonymous mutation in a conserved site of the MTTP gene is strongly associated with protein activity and fatty acid profile in pigs

Despite the economic interest of the fatty acid profile in pigs, no gene has been convincingly associated with this trait so far. Here, the porcine microsomal triglyceride transfer protein ( MTTP ) gene, which plays a crucial role in the assembly of nascent lipoproteins, has been analysed as a positional candidate gene for a QTL affecting the fatty acid composition that was previously identified on chromosome 8 in an Iberian by Landrace F₂ cross. By resequencing a panel of different breeds, a non-synonymous polymorphism in a conserved residue of the lipid transfer domain of MTTP was identified. Association analyses with this polymorphism showed a strong association with the fatty acid composition of porcine fat, much stronger than the QTL effect, in the F₂ cross and in a synthetic Sino-European line. In addition, in vitro activity assays in liver protein extracts have shown that this mutation is also associated with the lipid transfer activity of the MTTP protein ( P  <   0.1). These results suggest that the detected polymorphism is a potential causal factor of the fatty acid composition QTL. There appears to be an interaction between the porcine MTTP genotype and the type of fat source in the pig diet, which would agree with the previous results on the biology of MTTP biology.