Genetic and functional confirmation of the causality of the DGAT1 K232A quantitative trait nucleotide in affecting milk yield and composition

Abstract

We recently used a positional cloning approach to identify a nonconservative lysine to alanine substitution (K232A) in the bovine DGAT1 gene that was proposed to be the causative quantitative trait nucleotide underlying a quantitative trait locus (QTL) affecting milk fat composition, previously mapped to the centromeric end of bovine chromosome 14. We herein generate genetic and functional data that confirm the causality of the DGAT1 K232A mutation. We have constructed a high-density single-nucleotide polymorphism map of the 3.8-centimorgan BULGE30 -BULGE9 interval containing the QTL and show that the association with milk fat percentage maximizes at the DGAT1 gene. We provide evidence that the K allele has undergone a selective sweep. By using a baculovirus expression system, we have expressed both DGAT1 alleles in Sf9 cells and show that the K allele, causing an increase in milk fat percentage in the live animal, is characterized by a higher Vmax in producing triglycerides than the A allele.A quantitative trait locus (QTL) with major effect on milk fat composition has been mapped to the centromeric end of bovine chromosome 14 (1, 2). Linkage disequilibrium (LD) was used to refine the map position of the QTL to a 3.8-centimorgan interval bounded by microsatellite markers BULGE30 and BULGE9 (3, 4). A bacterial artificial chromosome (BAC) contig spanning this interval was constructed and shown to contain a very strong positional candidate: diacylglycerol acyl transferase 1 (DGAT1) (5). DGAT1 indeed catalyzes the last step in triglyceride synthesis (6) and abrogates milk yield when knocked out in the mouse (7). By sequencing the DGAT1 gene from individuals with known QTL genotype, a nonconservative lysine to alanine substitution was identified at position 232, and shown to be associated with a major effect on milk yield and composition in several dairy cattle populations and breeds (5,8,9). The DGAT1 K232A mutation was therefore considered to be the likely quantitative trait nucleotide underlying the BTA14 QTL effect.However, based on the available data, we could not formally exclude that the effect observed with the K232A polymorphism was, in fact, caused by another mutation, located in the same or in another gene mapping to the BULGE30-BULGE9 interval that would be in strong LD with K232A. To resolve this issue, we herein (i) describe the development of a high-density map of single-nucleotide polymorphisms (SNP) of the BULGE30-BULGE9 interval and show that the association with milk yield and composition is strongest for the DGAT1 SNPs, thereby strongly incriminating that gene; (ii) present evidence that the K allele has been under positive selection supporting its effect on the functionality of DGAT1; and (iii) demonstrate that the K232A mutation increases the activity of the enzyme in a way that is in agreement with its effect on phenotype.

The Breeding Values of German Holstein Sires and the <i>DGAT1</i> Polymorphism

“…According to HARDER et al (2006), the DGAT1 locus seems to be associated with fat yield throughout the whole lactation, and with milk yield and protein yield especially during the later stages of lactation. GRISART et al (2004) expressing both DGAT1 alleles in Sf9 cells using the baculovirus expression system, confirmed the DGAT1 K allele as being characterized by higher triglyceride production than the DGAT1 A . There is no single polymorphism in the respective chromosomal region; KÜHN et al (2004) evidence the DGAT1 promoter polymorphism affecting the fat content; BENNEWITZ et al (2004) presuppose an additional source of genetic variance on the bovine chromosome 14 and WINTER et al (2004) mapped twenty-three genes neighboring DGAT1, and report the respective polymorphisms.…”

Identification of a missense mutation in the bovine ABCG2 gene with a major effect on the QTL on chromosome 6 affecting milk yield and composition in Holstein cattle

“…However, as long as the entire chromosomal segment within the confidence interval of the QTL has not been sequenced in the sires with known QTL genotypes, it is not possible to completely eliminate the possibility that the QTN may be some other polymorphism in strong LD with Y581S. Furthermore, formal proof that this polymorphism is in fact the QTN can only be obtained by functional studies (Grisart et al 2004).…”

Fine-mapping milk production quantitative trait loci on BTA6: Analysis of the bovine osteopontin gene

“…A logarithm of odds (lod) score was obtained under the QTL and no-QTL models (y ϭ ϩ X␤ ϩ Zu ϩ e). Alternative analyses were also performed: (i) exploiting only LK information by ignoring all MH and regarding SH as distinct haplotype clusters (22) and (ii) marker association tests in which marker alleles were treated as haplotypes (28). lod score thresholds were determined at point-wise P levels by considering 2 ϫ ln (10) ϫ lod to be distributed as 1 2 (models differ by one parameter; H 2 ), because the QTL had previously been detected (29).…”