Detection of QTL for milk production traits in cattle by application of a specifically developed marker map of BTA6

Interval mapping was carried out to identify quantitative trait loci (QTL) for milk production traits in five granddaughter design families of the German Holstein population. Fourteen randomly generated markers spanning the whole of BTA6 and six targeted microsatellite markers from BTA6q21-31 were included in the analysis. In one family a QTL with effects on milk fat yield and milk protein yield was mapped to the interval TGLA37-FBN13 (3 CM proximal to FBN13, lodscore 3.22) in the middle part of the chromosome. Although there are several reports about QTL with effects on milk production traits on BTA6 in the literature, a QTL with effects on milk fat and milk protein yield has not been previously described.     

Quantitative trait locus-by-environment interaction for milk yield traits on Bos taurus autosome 6

Genotype-by-environment interactions for production traits in dairy cattle have often been observed, while QTL analyses have focused on detecting genes with general effects on production traits. In this study, a QTL search for genes with environmental interaction for the traits milk yield, protein yield, and fat yield were performed on Bos taurus autosome 6 (BTA6), also including information about the previously investigated candidate genes ABCG2 and OPN. The animals in the study were Norwegian Red. Eighteen grandsires and 716 sires were genotyped for 362 markers on BTA6. Every marker bracket was regarded as a putative QTL position. The effects of the candidate genes and the putative QTL were modeled as a regression on an environmental parameter (herd year), which is based on the predicted herd-year effect for the trait. Two QTL were found to have environmentally dependent effects on milk yield. These QTL were located 3.6 cM upstream and 9.1 cM downstream from ABCG2. No environmentally dependent QTL was found to significantly affect protein or fat yield.     

Genome-wide association study identifies two major loci affecting calving ease and growth-related traits in cattle

Identifying quantitative trait loci (QTL) underlying complex, low-heritability traits is notoriously difficult. Prototypical for such traits, calving ease is an important breeding objective of cattle (Bos taurus)-improving programs. To identify QTL underlying calving ease, we performed a genome-wide association study using estimated breeding values (EBVs) as highly heritable phenotypes for paternal calving ease (pCE) and related traits. The massively structured study population consisted of 1800 bulls of the German Fleckvieh (FV) breed. Two pCE-associated regions on bovine chromosomes (BTA) 14 and 21 (P = 5.72 × 10(-15) and P = 2.27 × 10(-8), respectively) were identified using principal components analysis to correct for population stratification. The two most significantly associated SNPs explain 10% of the EBV variation. Since marker alleles with negative effect on pCE have positive effects on growth-related traits, the QTL may exert their effects on the birthing process through fetal growth traits. The QTL region on BTA14 corresponds to a human chromosome (HSA) region that is associated with growth characteristics. The HSA region corresponding to the BTA21 pCE QTL is maternally imprinted and involved in the Prader-Willi and Angelman syndromes. Resequencing of positional candidate genes on BTA14 revealed a highly significantly (P = 1.96 × 10(-14)) associated polymorphism ablating a polyadenylation signal of the gene encoding ribosomal protein S20 (RPS20). Our study demonstrates the leverage potential of EBVs in unraveling the genetic architecture of lowly heritable traits.     

A candidate gene association study for nine economically important traits in Italian Holstein cattle

We genotyped 58 single nucleotide polymorphisms (SNPs) in 25 candidate genes in about 800 Italian Holstein sires. Fifty‐six (minor allele frequency >0.02) were used to evaluate their association with single traits: milk yield (MY), milk fat yield (FY), milk protein yield (PY), milk fat percentage (FP), milk protein percentage (PP), milk somatic cell count (MSCC); and complex indexes: longevity, fertility and productivity–functionality type (PFT), using deregressed proofs, after adjustment for familial relatedness. Thirty‐two SNPs were significantly associated (proportion of false positives <0.05) with different traits: 16 with MSCC, 15 with PY, 14 with MY, 12 with PFT, eight with longevity, eight with FY, eight with PP, five with FP and two with fertility. In particular, a SNP in the promoter region of the PRLR gene was associated with eight of nine traits. DGAT1 polymorphisms were highly associated with FP and FY. Casein gene markers were associated with several traits, confirming the role of the casein gene cluster in affecting milk yield, milk quality and health traits. Other SNPs in genes located on chromosome 6 were associated with PY, PP, PFT, MY (PPARGC1A) and MSCC (KIT). This latter association may suggest a biological link between the degree of piebaldism in Holstein and immunological functions affecting somatic cell count and mastitis resistance. Other significant SNPs were in the ACACA, CRH, CXCR1, FASN, GH1, LEP, LGB (also known as PAEP), MFGE8, SRC, TG, THRSP and TPH1 genes. These results provide information that can complement QTL mapping and genome‐wide association studies in Holstein.     

Mapping of a milk production quantitative trait locus to a 1.056 Mb region on bovine chromosome 5 in the Fleckvieh dual purpose cattle breed

Background

In a previous study in the Fleckvieh dual purpose cattle breed, we mapped a quantitative trait locus (QTL) affecting milk yield (MY1), milk protein yield (PY1) and milk fat yield (FY1) during first lactation to the distal part of bovine chromosome 5 (BTA5), but the confidence interval was too large for positional cloning of the causal gene. Our objective here was to refine the position of this QTL and to define the candidate region for high-throughput sequencing.

Methods

In addition to those previously studied, new Fleckvieh families were genotyped, in order to increase the number of recombination events. Twelve new microsatellites and 240 SNP markers covering the most likely QTL region on BTA5 were analysed. Based on haplotype analysis performed in this complex pedigree, families segregating for the low frequency allele of this QTL (minor allele) were selected. Single- and multiple-QTL analyses using combined linkage and linkage disequilibrium methods were performed.

Results

Single nucleotide polymorphism haplotype analyses on representative family sires and their ancestors revealed that the haplotype carrying the minor QTL allele is rare and most probably originates from a unique ancestor in the mapping population. Analyses of different subsets of families, created according to the results of haplotype analysis and availability of SNP and microsatellite data, refined the previously detected QTL affecting MY1 and PY1 to a region ranging from 117.962 Mb to 119.018 Mb (1.056 Mb) on BTA5. However, the possibility of a second QTL affecting only PY1 at 122.115 Mb was not ruled out.

Conclusion

This study demonstrates that targeting families segregating for a less frequent QTL allele is a useful method. It improves the mapping resolution of the QTL, which is due to the division of the mapping population based on the results of the haplotype analysis and to the increased frequency of the minor allele in the families. Consequently, we succeeded in refining the region containing the previously detected QTL to 1 Mb on BTA5. This candidate region contains 27 genes with unknown or partially known function(s) and is small enough for high-throughput sequencing, which will allow future detailed analyses of candidate genes.     

Investigation of the genetic architecture of a bone carcass weight QTL on BTA6

A previous analysis of an F₂/Backcross Charolais × Holstein cross population identified the presence of a highly significant QTL on chromosome 6 (BTA6) affecting the proportion of bone in the carcass. Two closely linked QTL affected birth weight (BW) and body length at birth (BBL). In this report, the marker density around the QTL on BTA6 was increased, adding four additional microsatellite markers across the chromosome and 46 SNPs within the target QTL confidence interval. Of the SNPs, 26 were in positional candidate genes and the remaining 20 provided an even distribution of markers in the target QTL region. As a bone‐related trait, the sum of the bone weight for all the left fore‐ and hindquarter joints of the carcass was analysed. We also studied the BW and BBL. Analyses of the data substantially reduced the QTL confidence interval. No strong evidence was found that the QTL for the three traits studied are different, and we conclude that the results are consistent with a single pleiotropic QTL influencing the three traits, with the largest effects on the proportion of bone in the carcass. The analyses also suggest that none of the SNPs tested is the sole causative variant of the QTL effects. Specifically, the SNP in the NCAPG gene previously reported as a causal mutation for foetal growth and carcass traits in other cattle populations was excluded as the causal mutation for the QTL reported here. Polymorphisms located in other previously identified candidate genes including SPP1, ABCG2, IBSP, MEPE and PPARGC1A were also excluded. The results suggest that SNP51_BTA‐119876 is the polymorphism in strongest linkage disequilibrium with the causal mutation(s). Further research is required to identify the causal variant(s) associated with this bone‐related QTL.     

Conflicting candidates for cattle QTLs

Genome scans have identified quantitative trait loci (QTLs) affecting milk yield and composition in dairy cattle. For one QTL on bovine chromosome 6 (BTA6), previously fine-mapped to a 420-Kb region, mutations in two different genes (OPN and ABCG2) have been proposed as the underlying functional mutation. Comparing the arguments for each gene suggests that both mutations are equally probable. However, functional studies and/or additional populations are required to provide a definite answer.     

中国荷斯坦牛CVM的基因检测及其与产奶性状的关联分析

脊椎畸形综合征(Complex vertebral malformation, CVM)是由位于牛第3号染色体(BTA3)的SLC35A3基因外显子4的一个单碱基突变(G559T)所致。该致病基因在世界许多国家的荷斯坦牛群中都有一定的比例。文章对北京地区38头优秀种公牛进行分析, 发现了4头携带者, 进而检测了这些携带者公牛的555头女儿的基因型, 其中携带者占检测母牛数的44.0%。此外, 关联分析结果表明, 携带者母牛与非携带者母牛的生产性能之间存在显著差异(P<0.01)。携带者母牛的5个产奶性状育种值均显著高于非携带者, 泌乳持续力和体细胞评分SCS的育种值也比非携带者略高。CVM致病基因可能与BTA3上影响产奶性状的QTL或基因连锁。因此, 建议生产中对CVM携带者进行逐步淘汰     

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.     

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.     

Identification of single nucleotide polymorphisms in the bovine CCL2, IL8, CCR2 and IL8RA genes and their association with health and production in Canadian Holsteins

The aim of this study was to identify the presence of SNPs in the chemokine genes CCL2 and IL8 and the chemokine receptor genes IL8RA and CCR2, and assess their potential contribution to variation in estimated breeding values (EBVs) for somatic cell score (SCS) and four other traits in Canadian Holstein bulls. Pools of DNA for bulls with high (H) and low (L) EBVs for SCS were used for identification of 11 SNPs. Two unreported SNPs were found in the CCL2 gene and one SNP was found in the CCR2 gene. Previously reported SNPs (three in the IL8 gene and five in the IL8RA chemokine receptor) were also identified. Two SNPs in CCL2, three in IL8, one in IL8RA and one in CCR2 were genotyped in Canadian Holstein bulls (n = 338) using tetra primer ARMS-PCR. We investigated associations of these seven polymorphisms with three production traits (milk yield, fat yield and protein yield) and one conformation trait related to mastitis (udder depth). The allele substitution effect for the CCL2 rs41255713:T>C SNP was significant at an experimental-wise level for milk yield (247.5 +/- 79.9 kg) and protein yield (7.4 +/- 2.3 kg) EBVs (P T SNP on SCS was significant at the comparison-wise level (-0.04 +/- 0.02, P = 0.05), which might indicate a possible association in support of other published studies. Lastly, we assigned CCR2 to BTA22q24, where a previously QTL for SCS was identified.     

The role of bovine causal genes underlying dairy traits in Spanish Churra sheep

In dairy cattle, quantitative trait nucleotides (QTNs) underlying quantitative trait loci (QTL) for milk production traits have been identified in bovine DGAT1, GHR and ABCG2 genes. The SPP1 gene has also been proposed to be a regulator of lactation. In sheep, QTL underlying milk production traits have been reported only recently, and no proven QTN has been identified. Taking into account the close phylogenetic relationship between sheep and cattle, this study examined the possible effects of the aforementioned genes on sheep milk production traits. We first studied the genetic variability of the DGAT1, GHR, ABCG2 and SPP1 genes in 15 rams of the Spanish Churra dairy sheep breed. Second, we performed an association analysis between SNPs identified in these genes and three milk production traits recorded in a commercial population of Churra sheep. This analysis revealed only three significant associations at the nominal level (P-value <0.05) involving allelic variants of the ABCG2 gene, whereas no significant association was found for the DGAT1, GHR and SPP1 genes. When the Bonferroni correction was applied to take into account the multiple tests performed, none of the associations identified at the nominal level remained significant. Nevertheless, taking into account the high level of false-negative findings that can arise when applying the stringent Bonferroni correction, we think that our results provide a valuable primary assessment of strong candidate genes for milk traits in sheep.     

The SNP c.1326T>G in the non-SMC condensin I complex, subunit G (NCAPG) gene encoding a p.Ile442Met variant is associated with an increase in body frame size at puberty in cattle

Recently, we had located a bovine carcass weight QTL, CW-2, to a 591-kb interval on BTA6 and have identified the SNP c.1326T>G in the NCAPG (non-SMC condensin I complex, subunit G) gene that leads to the amino acid change p.Ile442Met in the NCAPG protein, which is a candidate causative variation. Here, we examined the association of the NCAPG:c.1326T>G locus with linear skeletal measurements of growth-associated traits during adolescence, which is a period of intensive growth, using two historically and geographically distant cattle populations: 792 Japanese Black steers and 161 F(2) bulls of an experimental cross from Charolais and German Holstein. In both populations, the SNP NCAPG:c.1326T>G was associated with each component of body frame size: height, length and width at puberty. The associations of CW-2 with height- and length-associated traits were observed at an earlier growth period compared to the associations with thickness- and width-associated traits, indicating that the primary effect of the CW-2 QTL may possibly be exerted on skeletal growth. The significant associations of the NCAPG:c.1326T>G locus with growth-associated skeletal measurements are similar to the effects of the syntenic region on human chromosome 4 that are associated with adult height in humans, supporting the hypothesis that CW-2 is analogous to the human locus and pointing to a conserved growth-associated locus or chromosomal region present in both species.     

Novel SNPs in the bovine ADIPOQ and PPARGC1A genes are associated with carcass traits in Hanwoo (Korean cattle)

Adiponectin (ADIPOQ) modulates several biological processes including energy homeostasis, glucose and lipid metabolism. The bovine ADIPOQ gene was located near the QTL affecting marbling, ribeye muscle area and fat thickness on BTA1. The gene encoding peroxisome proliferator-activated receptor-γ coactivator-1α (PPARGC1A) was located within the QTL region of the traits on BTA6. Moreover, its protein product has various biological functions such as cellular energy homeostasis, including adaptive thermogenesis, adipogenesis and gluconeogenesis. Therefore, the ADIPOQ and PPARGC1A genes are a positional and functional candidate gene for carcass traits in beef cattle. The objectives of this study were to identify polymorphisms in the bovine ADIPOQ and PPARGC1A genes, to evaluate their associations with carcass traits in Hanwoo (Korean cattle) population. We identified nine SNPs in the ADIPOQ gene. Two SNPs (DQ156119: g.1436T > C and DQ156119: g.1454A > G) in the promoter region were recognized as new SNPs identified in Hanwoo. Association analysis indicated that the g.1454A > G SNP genotype was significantly associated with effects on LMA (P = 0.004) and BF (P = 0.021). The ADIPOQ haplotype was also found to have significant effect on the LMA. In the PPARGC1A gene, we identified 11 SNPs in the two unexplored regions (intron 3 and 5). Among them, seven SNPs were located in intron 3 and four SNPs were located in intron 5. Of these 11 putative novel SNPs, two SNPs (AY839822: g.292C > T and AY839823: g.1064C > T) with minor allele frequency (MAF) > 0.20 were examined for associations with carcass traits. The association analysis revealed that both SNPs in PPARGC1A gene were significantly associated with LMA (P < 0.05). These findings suggest that the SNPs of bovine ADIPOQ and PPARGC1A genes may be a useful molecular marker for selection of carcass traits in Hanwoo.     

Genomic organization of the DGAT2/MOGAT gene family in cattle (Bos taurus) and other mammals

We report the cloning and initial characterization of the genes encoding DGAT2 (diacylglycerol transferase 2), MOGAT1 and MOGAT2 (monoacylglycerol transferases 1 and 2) in domestic cattle (Bos taurus). The three closely related genes belong to a gene family with at least eight members in mammals and are candidate genes for quantitative traits related to dietary fat uptake, lipid synthesis and storage. MOGAT2 and DGAT2 form a tandem and were mapped to bovine chromosome (BTA) 15q25-->q26 by fluorescence in situ hybridization. MOGAT1 was localized to BTA 2q43-->q44. The three genes were investigated for polymorphisms that might be associated with breeding values for milk fat percentage in the dairy breeds German Holstein, German Simmental and German Brown. All the detected polymorphisms were located outside exons or, with one exception, were silent. In MOGAT1, a missense mutation in exon 4 was found that causes a non-conservative substitution of cysteine170 (uncharged, hydrophobic) by lysine (positively charged, hydrophilic). However, allele frequency estimates from pooled DNA samples revealed no significant association of the observed polymorphisms with breeding values for milk fat percentage. A comparative analysis of chromosomal locations and exon-intron structure of the known members of the DGAT2/MOGAT gene family in humans, rodents and cattle indicates an ancient tandem duplication of the ancestor gene combined with an intron gain (or loss) in one copy. Further members of the family may have arisen by duplications of this gene tandem via two rounds of interchromosomal or genome duplications as well as further local (single) gene duplication and loss events.     

Association between CSN3 and BCO2 gene polymorphisms and milk performance traits in the Czech Fleckvieh cattle breed

Daily milk, fat and protein yield and amount of somatic cells in cow milk are very important factors that influence milk performance traits. An association between polymorphisms in the kappa casein (CSN3) gene and milk production, composition and technical properties has been previously reported; however, this type of information is not available for the bovine β-carotene oxygenase 2 (BCO2) gene--the BCO2 gene has relationship with milk color and meat fat color, which is dependent on content of β-carotene. We analyzed these two genes and their relationship with milk performance traits (daily milk, fat and protein yield, somatic cell count, SCC) in one cattle population, Czech Fleckvieh (N = 152). All animals were milked twice a day and kept in the same environmental conditions. The Fleckvieh is a typical Czech cattle breed farming for milk and meat production. It is the most common breed in the Czech Republic. DNA was isolated from milk or from hairs. Genes were analyzed using PCR-RFLP, frequencies of alleles and genotypes were calculated and association analysis was performed using a GLM Procedure in SAS. Statistical analysis established that the CSN3 gene has no statistically significant influence on daily milk, fat and protein yield and SCC. Compared to other references this result can be explained by, e.g., small group of animals and different cattle breed. The BCO2 gene (genotypes AA and AG) shows a statistically significant relationship (P = 0.05) with daily milk, protein yield and SCC.     

Effect ofABCG2,PPARGC1A,OLR1 andSCD1 gene polymorphism on estimated breeding values for functional and production traits in Polish Holstein-Friesian bulls

This study investigated the impact of 6 polymorphisms located in theABCG2, PPARGC1A, OLR1 andSCD1 genes on estimated breeding values for milk production, longevity, somatic cell count and reproductive traits. The analysis was conducted on 453 Polish Holstein-Friesian bulls. Genotypes were identified using PCR-RFLP, and haplotype inferences were performed for 3 linked mutations ofPPARGC1A. The most significant associations were found between the A/C polymorphism located in exon 14 ofABCG2 and milk fat production traits as well as calving-to-first insemination interval, and between the T/C substitution in intron 9 of thePPARGC1A and non-return rate in heifers.     

Polymorphism identification, RH mapping and association of placental lactogen gene with milk production traits of dairy cows

Bovine placental lactogen (bPL) is structurally related to prolactin (PRL) and growth hormone (GH). In synergism with steroid and thyroid hormones, bPL is crucial in stimulating the development of the mammary gland, mammary cell differentiation and function. To further explore whether bPL gene is associated with milk production traits, we herein analyzed single-nucleotide polymorphisms (SNPs) within eight regions of bPL gene, which are potentially associated with five milk production traits on 1028 Chinese Holstein cows. Among these, two SNPs, NT7409(T-C) and Nt11246(G-A), were identified. The former is within exon 2; it induces an alteration of amino acid from Val to Ala. The later is within exon 4. It is a synonymous mutation. We found that there were significant associations between NT7409(T-C) and milk and protein yield. Cows of the AA genotype yielded less milk (P = 0.001) and less protein (P = 0.003) than those of genotypes AB and BB. However, on the NT11246(G-A) locus, no significant association was observed in the five milk production traits studied. In addition, bPL has been localized near markers RM185 and CC549051 with a distance of 23.2 cR on BTA 23. It is at the same position as the region including quantitative trait loci (QTLs) affecting milk and protein yields by previous linkage analysis. In summary, our findings demonstrated that the SNP within exon 2 of bPL (NT7409(T-C)) is associated with two milk production traits, and this provided further evidence that bPL could be a major gene-controlling milk production trait in Holstein dairy cattle.