Characterizing a region on BTA11 affecting β-lactoglobulin content of milk using high-density genotyping and haplotype grouping
© The Author(s). 2017
Received: 9 September 2016
Accepted: 11 February 2017
Published: 22 February 2017
Milk β-lactoglobulin (β-LG) content is of interest as it is associated with nutritional and manufacturing properties. It is known that milk β-LG content is strongly affected by genetic factors. In cattle, most of the genetic differences are associated with a chromosomal region on BTA11, which contains the β-LG gene. The aim of this study was to characterize this region using 777 k SNP data (BovineHDbeadChip) and perform a haplotype-based association study. A statistical approach was developed to build haplotypes that capture the genetic variation associated with this genomic region.
The SNP with the most significant effect on β-lactoglobulin content was one of the 2 causal mutations responsible for the β-lactoglobulin protein variants A/B. Haplotypes based on 2 to 5 selected lead SNP were clustered in groups with different effects on β-lactoglobulin content. Four different groups were identified suggesting that β-lactoglobulin variant A and B can be further refined in A1, A2, B1 and B2.
This study showed that β-lactoglobulin protein variants A/B do not explain all genetic variation associated with the tail part of BTA11 but this region contains more than one mutation with an effect on β-lactoglobulin content. These findings can be used for selection of cows with higher cheese yield, which is desirable for the dairy industry.
KeywordsDairy cow Bovine β-lactoglobulin Haplotype Association study
Bovine milk contains around 3–4% protein, which consists of caseins and whey proteins. Around 80% of the milk proteins are caseins and the remaining fraction is made up of soluble proteins of which β-lactoglobulin (β-LG) is the most important [1, 2]. β-LG is of interest as it is associated with nutritional and manufacturing properties of milk. Interestingly, human milk does not contain β-LG and, therefore, β-LG may be less important for human infants. Some people are oversensitive to milk protein (cow’s milk allergy) and β-LG has been considered as a major milk allergen . This was one of the reasons for selecting a cow with milk lacking β-LG . On the other hand, β-LG is a rich source of essential amino acids and has therefore a high nutritional value .
Two distinct forms of the β-LG protein (A and B) were described in 1955  and several studies have shown relations between protein variants A and B of β-LG, cheese yield and heat stability of milk [7, 8]. Milk from cows homozygous for β-LG protein variant B results in approximately 3% more cheese as compared to milk from cows homozygous for β-LG protein variant A . Further, milk with β-LG protein variant B results in a lower fouling rate of heating equipment  and therefore in lower costs of cleaning heating equipment.
Milk β-LG content is strongly affected by genetic factors: 80% of the differences are due to genetics . A genome wide association study identified a chromosomal region on BTA11 with a major effect on β-LG content . This region contains the β-LG gene which codes for the β-LG protein. Several studies showed that β-LG protein variants A and B are associated with β-LG content in the milk: the β-LG B variant is associated with a lower β-LG content [12–14]. Schopen et al.  found that after adjusting for the effects of β-LG protein variants a significant proportion of the genetic variance remains associated with the chromosomal region on BTA11. This suggests that the mutations responsible for the differences between β-LG A/B protein variants are not the causal mutations or that this region contains multiple mutations with an effect on β-LG content. The recent availability of high density (777 k) SNP array enables to fine map the targeted region on BTA11 and investigate if one or multiple mutations are responsible for the observed effects. In addition, defining haplotypes that capture all genetic variation in β-LG content associated with this region will allow more efficient selection for β-LG content than would be possible based on β-LG protein variants.
This study aims to fine map the chromosomal region on BTA11 associated with β-LG content using 777 k SNP data and to investigate if one or multiple mutations are responsible for the observed effects.
Variance components (herd variation, polygenic additive genetic variation and residual variation), intra-herd heritability and proportion of variance due to herd for the un-adjusted β-LG content (wt/wt%) and the adjusted β-LG contents
Un-adjusted β-LG content
Adjusted β-LG content
Single SNP association
Information on the Q-Tag SNP identified in this study
Position (based on Btau 4.2)
Variance components (additive genetic variation, haplo-group variation and residual variation), proportion of variance due to haplo-groupsa and estimated effects of haplo-groups on β-LG content and their distribution in the population
Haplotype based on
2 Q-Tag SNP
3 Q-Tag SNP
4 Q-Tag SNP
5 Q-Tag SNP
haplo-group A1 a
When considering Q-Tag SNP1 and Q-Tag SNP2, haplotypes GG, GA, AG and AA can be distinguished. Pairwise comparisons of the predicted haplotype effects show that all four haplotypes have significantly different effects on β-LG content. Haplotype GG has the strongest positive effect on β-LG content and its effect is significantly different from haplotype GA, which also is associated with an increase of β-LG content. The GG and the GA haplotypes differentiate among β-LG protein variant A and will therefore be referred to as A1 (GG) and A2 (GA). The two other haplotypes AG and AA differentiate β-LG protein variant B and will be referred to as B2 (AG) and B1 (AA). These two haplotypes are associated with lower β-LG content. The estimated difference between haplotype GG and AA is 1.60% and therefore the expected difference between cows carrying two copies of haplotype GG versus those who have two copies of haplotypes AA is 3.20%.
Further refining the haplotypes by considering three Q-Tag SNPs results in one haplotype (AGA) which occurs at a very low frequency in the population (1%). Adding the third Q-Tag SNP does result in a further refinement: the GG haplotype, which was assigned to haplo-group A1, is differentiated in AGG and GGG haplotypes which have significantly different effects. Whereas the AGG haplotype is assigned to haplo-group A1, the GGG haplotype is assigned to haplo-group A2.
When considering 4 or 5 Q-Tag SNP to build haplotypes, there is an increasing number of haplotypes that occur at low frequencies in the population. The predicted effects of haplotypes with frequencies smaller than 1% were not included in Fig. 2. Haplotypes consisting of 4 or more Q-Tag SNP could not always be unequivocally assigned to one of the four haplo-groups, e.g. the predicted effect of haplotype AGAC (haplo-group A2) is not significantly different from effects of haplotypes in haplo-group A1 and the predicted effect of haplotype AAAC (haplo-group B1) is not significantly different from effects of haplotypes in haplo-group B2.
Table 3 shows the estimated variance components, genetic parameters and estimated effects for the haplo-groups. Haplotypes were assigned to one of the four haplo-groups as is shown in Fig. 2. For comparison, results are also shown for a situation when considering only one Q-Tag SNP, i.e. modelling the allelic effects of the β-LG protein variants as a random effect. Results show that haplotype variance increases from 0.664 to 0.685 when moving from 2 to 3 Q-Tag SNP whereas the residual polygenic additive genetic variation tends to decrease (0.297–0.293). Adding more than 3 Q-Tag SNP did not further increase the variance explained by the haplo-groups. The proportion of the variance explained by differences among haplo-groups was 63.7% when considering 2 Q-Tag SNP and increased to 64.5% when haplotypes were based on 3 or more Q-Tag SNP. In addition, the difference of estimated effect size on β-LG content between individuals homozygous for haplo-group A1 (A1A1) and individuals homozygous for haplo-group B1 (B1B1) increased from 2.86 for considering only one Q-Tag SNP to 3.26 when considering 3 or more Q-Tag SNP. The analyses indicate that 89% of the additive genetic variation in β-LG content can be explained by the genomic region between 100 Mb and 110 Mb of BTA11.
β-LG is a milk protein which is the product of the PAEP gene. Therefore it is expected that the phenotype-genotype relationship of milk β-LG content is relatively simple. The heritability of milk β-LG content was estimated to be 0.80 indicating that differences in β-LG content are strongly determined by genetic factors . A genome wide association study indicated that a chromosomal region on BTA11 containing PAEP explains most of the genetic variation in β-LG content . However, after adjusting for β-LG protein variants, a significant proportion of the genetic variance remains associated with this genomic region. The authors found another SNP that significantly explained 1.5% of the genetic variance in the region after adjusting for the effect for protein variants. This suggests that mutations responsible for the differences between β-LG A/B protein variants are either not causal or that there are multiple mutations in this chromosomal region with an effect on β-LG content. In the current study we defined haplotypes based on Q-Tag SNP and using this approach the genetic variation associated with a chromosomal region can be captured based on a relatively small number of SNP. The haplotypes were clustered in 4 groups, A1, A2, B1 and B2, with distinct effects on β-LG content suggesting that this chromosomal region contains more than one mutation with an effect on β-LG content.
Fine mapping using 777 k SNP array
Fine mapping the genomic region between 75 and 110 Mb on BTA11 using the high density SNP array (777 k) resulted in a substantial increase in SNP density as compared to the 50 k array SNP panel. Therefore, the high density SNP array is expected to increase the probability of finding SNP in strong Linkage Disequilibrium (LD) with the causal mutation(s). However, the lead SNP based on the 777 k array (Q-Tag SNP1) is identical to the lead SNP based on the 50 k array . Q-Tag SNP1 is one of the 2 causal mutations for β-LG protein variants A/B . Several studies, in different breeds and populations, have consistently shown associations between β-LG protein variant A and increased β-LG content [12, 14, 16]. This suggests that Q-Tag SNP1 actually may be one of the causal mutations or at least located close to the causal mutation. Q-Tag SNP1 explains most but not all of the additive genetic variation associated with this genomic region. This suggests that either the causal mutation has not been identified or that this region contains multiple mutations with an effect on β-LG content.
Haplotype construction and associations
The use of haplotypes in genome-wide association studies has been suggested because they may be in stronger LD with the Quantitative Trait Loci (QTL) than single SNP and therefore may have increased power to detect QTL [17, 18]. The advantage of haplotype over single SNP association study is expected to be smaller for high density as compared to low density SNP arrays. However, QTL with low Minor Allele Frequencies (MAFs) may be in low LD with SNP present on the SNP array due to ascertainment bias. In addition, single SNP may not be able to capture all genetic variation associated with a genomic region, e.g. because a region contains multiple causative mutations. Haplotypes provide a more detailed characterization of a region and can be used for dissecting effects associated with a genomic region.
An important difficulty with a haplotype-based approach is that the number of haplotypes becomes very large when haplotypes are based on an increasing number of SNP. E.g. when haplotypes are constructed based on the lead SNP and 10 adjacent SNP (5 on each side) 13 haplotypes are segregating in the current data and when 20 adjacent SNP are used (10 on each side of the lead SNP) the number of haplotypes is 53. Having a large number of haplotypes reduces the number of observations per haplotype: several haplotypes have frequencies smaller than 0.1%. The small number of observations per haplotype will likely dilute association signals. Construction haplotypes based on Q-Tag SNP strongly limits the number of possible haplotypes while still capturing the variation associated with a region. However, even when building haplotypes on Q-Tag SNP, the number of haplotypes is 2n where n is the number of Q-Tag SNP.
Haplotypes can be considered as alleles of a single multi-allelic marker and as such can be used in an association. The maximum number of genotype effects of this “super” marker is ½ m(m + 1) where m is the number of haplotypes (or alleles of the “super” marker). For example, for 8 haplotypes there are at maximum 36 effects to be estimated which is a further risk of diluting association signals. Therefore, we restricted the number of effects to be estimated by assuming additivity of the haplotype effects. The design matrices of both haplotypes were combined and the statistical analysis results in one estimated haplotype effect.
Even when using the described approach, inevitably a few common and several rare haplotypes will appear when the number of Q-Tag SNP increases (Fig. 2). These low frequency haplotypes may have a unique effect but it is not possible to significantly distinguish their effects from the effect other haplotypes. The current study shows that based on 3 Q-Tag SNP most of the additive genetic variation associated with this genomic region can be captured. Indeed, the additive genetic variation is about 1.121 for unadjusted β-LG content and 0.084 for haplotypes based on 3 Q-Tag SNP (i.e. a reduction of 93%). Any additional refining of haplotypes did not increase the genetic variation explained by the haplotypes. Adding Q-Tag SNP increases the number of haplotypes but in general decreases the number of cows carrying copies of a specific haplotype. This decreases the power of unequivocally assigning haplotypes to haplotype groups or to identify new haplotype groups with distinct effects.
Effects of haplotypes
In the current study we were able to identify 4 groups of haplotypes with distinct effects on β-LG content: A1, A2, B1 and B2. This is consistent with other study suggesting that the genetic variant A and B of PAEP can be further refined into 4 genetic variants in total through splitting both the A and B variants into 2 sub-variants . Both the SNPs identified in this study and the haplotypes constructed are different from the one of the present study although closely located and possibly linked with the same causal mutations. Effects of haplotypes at low frequency cannot be predicted very accurately and therefore complicates assigning them unequivocally to one of the existing haplo-groups. The results suggest that the number of haplotype groups with distinct effects does not increase beyond the already existing four when haplotypes are based on three Q-Tag SNPs. However, further refinement of the haplo-groups did take place: haplotype GG, which was assigned to haplo-group A1, was split in haplotype AGG which was assigned to haplo-group A1 and haplotype GGG which was assigned to haplo-group A2.
Having more than two groups of haplotypes suggests that this chromosomal region contains more than one mutation with an effect on β-LG content. Assuming that there are two mutations underlying the observed haplotype effects, i.e. locus 1 and locus 2, than haplo-group A1 carries a “+” allele at locus 1 and a “+” allele at locus 2, haplo-group A2 carries a “+”allele at locus 1 and a “-” allele at locus 2, haplo-group B2 carries a “-” allele at locus 1 and a “+” allele at locus 2 and haplo-group B1 carries a “-”allele at locus 1 and a “-” allele at locus 2. Using the results from Table 3 (based on 3 Q-Tag SNP), the estimated additive effect (i.e. “a” in Falconer notation) at locus 1 is 1.42 and 0.22 at locus 2. The estimated frequencies of the alleles which increase β-LG content are 0.58 at locus 1 and 0.66 at locus 2.
β-LG protein variants are not associated with protein content of milk but are strongly associated with the casein index . When analysing the haplo-groups, we also do not find an effect on milk protein content (h2 haplo-group = 0.00) but there is a large effect on the casein index (h2 haplo-group = 0.57). The estimated effect on the casein index is −0.87 for haplo-group A1, −0.69 for haplo-group A2, 0.62 for haplo-group B2 and 0.94 for haplo-group B1 (haplo-groups based on 3 Q-Tag SNP). The difference in casein index between the β-LG protein variants BB and AA is 3.15% whereas the difference between extreme haplotype groups (B1B1 vs. A1A1) is 3.63%. The casein index is directly related to the efficiency of cheese production and therefore selecting for B1B1 is beneficial to the dairy industry.
In order to find the causal mutations a possible next step is to sequence animals. The haplotypes can be used to design sequencing studies and individuals from different haplo-groups can be identified for sequencing (e.g. A1A1 versus B1B1). Although knowledge on the causal mutations is currently lacking, the identified haplotypes can be used in selection.
The lead SNP from the single SNP association using the high density SNP array is one of the 2 mutations responsible for the difference between β-LG protein variants A and B. The statistical approach developed can be used in fine mapping, haplotypes reconstruction and association studies with quantitative traits. A tool enabling to decide at which step to stop the stepwise association study has to be found. We constructed haplotypes based on 2 to 5 Q-Tag SNP and clustered in groups with significantly different effects on β-LG content. This study showed there are 4 different haplo-groups: A1, A2, B1 and B2 (named by analogy to protein variants A and B). The existence of more than two groups of haplotypes suggests that this chromosomal region contains more than one mutation with an effect on β-LG content. These findings can be used for selection of cows with higher cheese yield.
The present study was part of the Dutch Milk Genomics Initiative. In this project Milk samples were collected from 1,713 primiparous cows on 383 commercial herds. These cows descended from one of five proven bulls representing five large half-sib families (782 cows), one of 50 test bulls representing 50 small half-sib families (760 cows), or from 15 other proven bulls (171 cows). In the last group of 171 cows, at least 3 cows per herd were sampled. The pedigrees of the cows were supplied by the CRV (Arnhem, The Netherlands). Each cow was at least 87.5% Holstein-Friesian. The average age of the cows at first calving was 2.1 years and the cows calved between June 2004 and February 2005. Almost all the same animals were used in previous studies for the genetic analysis of milk protein [10, 11].
Morning milk samples, collected between February and March 2005 on 1,713 Dutch Holstein-Friesian cows, were analysed for detailed milk protein composition. The β-LG content was determined by Capillary Zone Electrophoresis (CZE) as described by Heck et al. (2008). Protein content (wt/wt%) was predicted based on infrared spectroscopy by routine milk recording (for more details see ).
DNA for genotyping was isolated from blood samples of 1,736 cows. A 50 k SNP chip developed by CRV (cooperative cattle improvement organization, Arnhem, the Netherlands) was used to genotype cows as well as the sires of the cows using the Infinium assay (Illumina, USA) . In addition, 55 of the sires of these cows were genotyped with the BovineHDbeadChip (about 777 k, Illumina, USA). For imputing the 1,736 cows from 50 to 777 k a reference population of 1,333 Dutch Holstein-Friesian cows was available. The reference population included the 55 sires. Other animals in the reference population were provided by CRV. For imputation and phasing BEAGLE 3.3 was used . In a first step, the consistency of genotypes between parents and offspring was assessed. The pedigree was assumed to be correct if less than 0.5% of the homozygous markers in the offspring were not in agreement with the parental genotype. In a second step, 777 k SNP genotypes were imputed and phased for all 1,736 cows using information of the 50 k SNP genotypes of all animals and the 777 k SNP genotypes of animals in the reference population .
The genotypes of the 2 SNP responsible for the amino acid changes in the β-LG variants A and B and 8 other SNP associated with β-LG content  were available for 1,611 cows. For 125 cows these SNP genotypes were missing and imputed and phased using BEAGLE 3.3 . The positions of the SNP were based on the Btau 4.2 assembly.
In total, 1,647 cows had both phenotypic and genotypic information and were used for the association study. Based on previous results , we focused on the region from 75 Mb to 110 Mb on BTA11 in the current study. In that region, 9,925 SNP genotypes were available of which 872 SNP were homozygous in our population and therefore not included in the association study.
where yklmnowas the β-LG content, μ is the mean for β-LG content, dimklmno is the covariate describing the effect of the numbers of days in milk, modelled with Wilmink curve  as explained in Heck et al. (2008) , caklmno is the covariate describing the effect of the age at first calving as linear and quadratic, seasonk is the fixed effect calving season (k = 1, 2 or 3), scodel is the fixed effect of sire group (l = 1, 2 or 3), SNPm is the fixed effect of the SNP, animaln is the random additive genetic effect of the animal n, herdo is the random herd effect and eklmno is the random residual effect. The animal effects were assumed to be distributed as N(0, A σ a 2 ), herd effects were assumed to be distributed as N(0, I σ herd 2 ) and the residuals were assumed to be distributed as N(0, I σ e 2 ), where A is the additive genetic relationships matrix, I is the identity matrix, σ a 2 is the additive genetic variance, σ herd 2 is the herd variance and, σ e 2 is the residual variance. The statistical package ASReml  was used to perform the analyses. In the association analysis, the variance components were fixed to estimates obtained from model (1) without the SNP effect.
where y* klmno is the β-LG content adjusted for the effect of the lead SNP genotype m. Estimated SNP genotype effects were obtained from model (1). Subsequently variance components were re-estimated for the adjusted phenotype (y* klmno) and the association study was repeated with variance components fixed at their new values. This procedure was repeated until P > 0.01 for the most significant SNP. The significant level of P-values = 0.01 equivalent to –log10(P-values) = 2 was chosen. False positive test were performed to check for multiple testing issue. In analogy to “Tag SNP”, i.e. a limited set of SNP that capture the genetic variation associated with a genomic region , we defined “Q-Tag SNP” as the set of SNP identified by the described procedure that capture the genetic variation of a chromosomal region. The β-LG content adjusted for the effect of Q-Tag SNP1 (lead SNP for the un-adjusted β-LG content) will be referred to as β-LG1, β-LG2 refers to β-LG1 adjusted for the effect of Q-Tag SNP2 (lead SNP for β-LG1) and so on.
where the variables are as described for model (1) with the SNP effect being replaced by haplotype effects haplo1o and haplo2p. haplo1o is the effect of the first copy of an animal’s haplotype and haplo2p is the effect of the second copy of an animal’s haplotype. The two haplotypes of an individual were randomly assigned to haplo1 or haplo2 and the design matrices of both haplotype effects were combined to estimate the effect of a particular haplotype. Haplotypes were modelled as random effects and assumed to be distributed as N(0, Iσ haplo 2 ) where I is the identity matrix and σ2 haplo is the variation due to haplotypes.
In order to determine the LD among the SNP between 75 Mb and 110 Mb of BTA11, the r2 was estimated using PLINK 1.07 . By default the software is unphasing the data but an optional command was used to keep the phasing information for calculation of LD .
Minor allele frequency
- PAEP :
Quantitative trait loci
Single nucleotide polymorphism
We would like to thank the owners of the herds for their help in collecting the data, the Milk Control Station (Zutphen, the Netherlands) for analysing the milk samples and CRV (Arnhem, the Netherlands) for supplying pedigrees and milk production data.
This study is part of the Milk Genomics Initiative, funded by Wageningen University, NZO (Dutch Dairy Organization), CRV (cooperative cattle improvement organization), and STW (Dutch technology foundation).
Availability of data and materials
Data are available upon request; contact Henk Bovenhuis by email: email@example.com. Part of these results were already made available and presented to the attendees of the 10th World Congress of Genetics Applied to Livestock Production (2014; Vancouver, BC Canada). The information was not published elsewhere; therefore the present paper is reporting an original research study.
NB carried out the analysis, prepared and drafted the manuscript. HB participated in the design of the study, the coordination of the study and drafting the manuscript. Both authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Genomic DNA of the cows was isolated from whole blood samples of the cows. Blood samples were collected in accordance with the guidelines for the care and use of animals as approved by the ethical committee on animal experiments of Wageningen University (protocol: 200523.b).
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