Impact of Variation at the FTO Locus on Milk Fat Yield in Holstein Dairy Cattle

<div><p>This study explores the biological role of the Fat Mass and Obesity associated (FTO) gene locus on milk composition in German Holstein cattle. Since FTO controls energy homeostasis and expenditure and the FTO locus has repeatedly shown association with obesity in human studies, we tested FTO as a candidate gene in particular for milk fat yield, which represents a high amount of energy secreted during lactation. The study was performed on 2,402 bulls and 860 cows where dense milk composition data were available. Genetic information was taken from a 2 Mb region around FTO. Five SNPs and two haplotype blocks in a 725 kb region covering FTO and the neighboring genes RPGRIP1L, U6ATAC, and 5 S rRNA were associated with milk fat yield and also affected protein yield in the same direction. Interestingly, higher frequency SNP alleles and haplotypes within the FTO gene increased milk fat and protein yields by up to 2.8 and 2.2 kg per lactation, respectively, while the most frequent haplotype in the upstream block covering exon 1 of FTO to exon 15 of RPGRIP1L had opposite effects with lower fat and milk yield. Both haplotype blocks were also significant in cows. The loci accounted for about 1% of the corresponding trait variance in the population. The association signals not only provided evidence for at least two causative mutations in the FTO locus with a functional effect on milk but also milk protein yield. The pleiotropic effects suggest a biological function on the usage of energy resources and the control of energy balance rather than directly affecting fat and protein synthesis. The identified effect of the obesity gene locus on milk energy content suggests an impact on infant nutrition by breast feeding in humans.</p></div

Bands excised from a mare with +/+-genotype (upper part) and Δ/Δ genotype (lower part), respectively, and major milk proteins identified are shown (only those with > 20 PSMs).

<p>AlphaS2-casein with accession no. A0A0C5DH76 is the long form (231 AA) while alphaS2-casein with accession no. D2KAS0 is the shorter form of alphaS2-casein (214 AAs). Unique peptides were only identified for the long form of alphaS2-casein (A0A0C5DH76) in the +/+-mare, while unique peptides for the short form of alhphaS2-casein (D2KAS0) were identified only in milk from the Δ/Δ-mare.</p

Frequencies of the 1.3 kb deletion in different horse breeds.

<p>^a ins = long variant corresponding to the genomic reference NC_009146.2; del = short variant encompassing the 1,339 bp deletion spanning two coding exons.</p><p>^b A synthetic cross involving German Riding Pony, Haflinger Horse, Connemara Pony and New Forrest Pony; bred for milk yield.</p><p>^c The frequency in Fjord horses is not reported with respect to the small sample size, but the breed is included in the total values.</p><p>Frequencies of the 1.3 kb deletion in different horse breeds.</p

Combined sequence coverage (all bands excised) of α_S2-casein in mare with +/+ genotype (A) and Δ/Δ genotype (B).

<p>From the +/+ genotype only unique peptides were identified for the longer α_S2-casein form (Accession no. A0A0C5DH76, 231 AAs). Sequence which is unique to A0A0C5DH76 is underlined. For the Δ/Δ genotype only unique peptides were identified for the shorter alphaS2-casein form (Accession no. D2KAS0, 214 AAs). Sections in green represents parts of the protein which were identified by the sequest-HT algorithm.</p

Structure of the long and short equine α_s2-casein variants.

<p>A. Genomic organization of the respective gene segment. Grey shading indicates the equid specific 309 bp duplication comprising coding exons 8 and 10, respectively. The 1.3 kb in-frame-deletion is indicated above the figure. B. Structures the resulting transcript variants. C. Protein alignment of available ungulate α_s2-casein protein sequences.</p

Identification and Dissection of Four Major QTL Affecting Milk Fat Content in the German Holstein-Friesian Population

<div><p>Milk composition traits exhibit a complex genetic architecture with a small number of major quantitative trait loci (QTL) explaining a large fraction of the genetic variation and numerous QTL with minor effects. In order to identify QTL for milk fat percentage (FP) in the German Holstein-Friesian (HF) population, a genome-wide association study (GWAS) was performed. The study population consisted of 2327 progeny-tested bulls. Genotypes were available for 44,280 SNPs. Phenotypes in the form of estimated breeding values (EBVs) for FP were used as highly heritable traits. A variance components-based approach was used to account for population stratification. The GWAS identified four major QTL regions explaining 46.18% of the FP EBV variance. Besides two previously known FP QTL on BTA14 (<em>P</em> = 8.91×10−¹⁹⁸) and BTA20 (<em>P</em> = 7.03×10⁻¹²) within <em>DGAT1</em> and <em>GHR</em>, respectively, we uncovered two additional QTL regions on BTA5 (<em>P</em> = 2.00×10⁻¹³) and BTA27 (<em>P</em> = 9.83×10⁻⁵) encompassing <em>EPS8</em> and <em>GPAT4</em>, respectively. <em>EPS8</em> and <em>GPAT4</em> are involved in lipid metabolism in mammals. Re-sequencing of <em>EPS8</em> and <em>GPAT4</em> revealed 50 polymorphisms. Genotypes for five of them were inferred for the entire study population. Two polymorphisms affecting potential transcription factor binding sites of <em>EPS8</em> (<em>P</em> = 1.40×10⁻¹²) and <em>GPAT4</em> (<em>P</em> = 5.18×10⁻⁵), respectively, were highly significantly associated with the FP EBV. Our results provide evidence that alteration of regulatory sites is an important aspect of genetic variation of complex traits in cattle.</p> </div

Agarose gel electrophoresis of equine <i>CSN1S2</i> cDNA.

<p>The RNA was isolated from the milk of a mare being homozygous for the deletion (Δ/Δ).and three mares homozygous for the long variant (+/+).</p

Agarose gel electrophoresis of PCR products spanning the 1.3 kb deletion.

<p>The upper visible band corresponds to the long variant (denoted as +), the lower one to the short variant containing the deletion (denoted as Δ). Only in heterozygotes, a third band with a size of approximately 2.1 kb is visible, which is possibly arising from asymmetric hybridization of the alleles due to the presence of a duplication. The breeds of the corresponding samples are given above the lanes (RHD = Russian Heavy Draft, GWb = German Warmblood, IC = Icelandic horse, HF = Haflinger).</p

Partitioning of the genetic variance onto 30 chromosomes and four identified QTL regions on BTA5, 14, 20 and 27.

<p>The grey shaded bars indicate the fraction of EBV variance attributed to a particular chromosome and the corresponding standard error. The dark grey bars represent the fraction of EBV variance attributed to each of the four identified QTL regions. The black triangles represent the cumulative proportion of EBV variance explained.</p

Characteristics of the most significantly associated 50 K Illumina BeadChip SNPs and additional polymorphisms of four major QTL for milk fat percentage in the German Holstein-Friesian population.

a<p>The SNPs are ordered according to their position on the UMD3.1 assembly of the bovine genome sequence.</p>b<p>The <i>P</i>-values are obtained after regression analysis and by using a variance components based approach to account for population stratification.</p