Casein SNP in Norwegian goats: additive and dominance effects on milk composition and quality

Background

The four casein proteins in goat milk are encoded by four closely linked casein loci (CSN1S1, CSN2, CSN1S2 and CSN3) within 250 kb on caprine chromosome 6. A deletion in exon 12 of CSN1S1, so far reported only in Norwegian goats, has been found at high frequency (0.73). Such a high frequency is difficult to explain because the national breeding goal selects against the variant''s effect.

Methods

In this study, 575 goats were genotyped for 38 Single Nucleotide Polymorphisms (SNP) located within the four casein genes. Milk production records of these goats were obtained from the Norwegian Dairy Goat Control. Test-day mixed models with additive and dominance fixed effects of single SNP were fitted in a model including polygenic effects.

Results

Significant additive effects of single SNP within CSN1S1 and CSN3 were found for fat % and protein %, milk yield and milk taste. The allele with the deletion showed additive and dominance effects on protein % and fat %, and overdominance effects on milk quantity (kg) and lactose %. At its current frequency, the observed dominance (overdominance) effects of the deletion allele reduced its substitution effect (and additive genetic variance available for selection) in the population substantially.

Conclusions

The selection pressure of conventional breeding on the allele with the deletion is limited due to the observed dominance (overdominance) effects. Inclusion of molecular information in the national breeding scheme will reduce the frequency of this deletion in the population.     

Variations at regulatory regions of the milk protein genes are associated with milk traits and coagulation properties in the Sarda sheep

Regulatory variation at the ovine casein genes could have important effects on the composition and coagulation properties of milk. Herewith, we have partially resequenced the promoters and the 3′‐UTR of the four casein genes in 25 Sarda sheep. Alignment of these sequences allowed us to identify a total of 29 SNPs. This level of polymorphism (one SNP every 250 bp) is remarkably high if compared with SNP densities estimated in human genic regions (approximately one SNP per bp). The 29 SNPs identified in our resequencing experiment, plus three previously reported SNPs mapping to the lactalbumin, alpha (LALBA) and β‐lactoglobulin (BLG, also known as progestagen‐associated endometrial protein, PAEP) genes, were genotyped with a multiplex TaqMan Open Array Real‐Time PCR assay in 760 Sarda sheep with records for milk composition and coagulation properties. Association analysis revealed the existence of significant associations of CSN1S2 and CSN3 genotypes with milk protein and casein contents. Moreover, genotypes at CSN1S1 were significantly associated with rennet coagulation time, curd firming time and curd firmness, whereas CSN2 was associated with curd firming time. These results suggest that SNPs mapping to the promoters and 3′‐UTRs of ovine casein genes may exert regulatory effects on gene expression and that they could be used for improving sheep milk quality and technological traits at the population level through marker assisted selection.     

Associations between 2 paternal casein haplotypes and milk yield traits of Swiss Fleckvieh cattle

Associations between casein haplotypes and milk yield traits of offspring from 5 Swiss Fleckvieh AI test bulls were investigated. The analysis was performed by using a daughter design, where each daughter inherited either paternal haplotype B-A1-A-A or B-A2-A-A for alleles of alpha s1-, beta-, alpha s2- and kappa-casein genes. The substitution effects of paternal CSN2 A1 versus A2 on protein yield deviations (YDs) were significant (P < 0.05), whereas their effects on milk and fat YDs were not. The paternal substitution effects of the CSN2 A1 versus the A2 allele on protein YDs within the 5 sires did not reach the significance level. This is due to the contrary allele substitution effect of a sire compared to the other 4 sires. The effects of maternal haplotypes on milk, protein and fat YDs were not significant. However, it is noteworthy that the effects of haplotypes with a low frequency in the population deviate largely from the most frequent haplotype B-A2-A-A. The effects of beta-lactoglobulin (BLG) genotypes were significant for protein YDs but not for milk and fat YDs. The association between the paternal CSN2 A1 and A2 alleles and milk protein YDs within sires but not milk and fat YDs indicate an interaction, which might be a consequence of CSN2 heterogeneity or a closely linked gene that is contributing to the estimated effects.     

Characterization of a QTL region affecting clinical mastitis and protein yield on BTA6

Quantitative trait loci affecting clinical mastitis were detected and fine mapped to a narrow region on bovine chromosome 6 in the Norwegian Red cattle population. The region includes the casein gene cluster and several candidate genes thought to influence clinical mastitis. The most significant results were found for SNPs within the Mucin 7 gene. This gene encodes an antimicrobial peptide and constitutes part of the first line of defence for the mucosal immune system. Detection of long haplotypes extending several Mb may indicate that artificial selection has influenced the haplotype structures in the region. A search for selection sweeps supports this observation and coincides with association results found both by single SNP and haplotype analyses. Our analyses identified haplotypes carrying quantitative trait loci alleles associated with high protein yield and simultaneously fewer incidences of clinical mastitis. The fact that such haplotypes are found in relative high frequencies in Norwegian Red may reflect the combined breeding goal that is characterized by selection for both milk production and disease resistance. The identification of these haplotypes raises the possibility of overcoming the unfavourable genetic correlation between these traits through haplotype-assisted selection.     

Goat casein genotypes are associated with milk production traits in the Sarda breed

The aim of the current work was to analyze, in the Sarda breed goat, genetic polymorphism within the casein genes and to assess their influence on milk traits. Genetic variants at the CSN1S1, CSN2, CSN1S2 and CSN3 gene loci were investigated using PCR‐based methods, cloning and sequencing. Strong alleles prevailed at the CSN1S1 gene locus and defective alleles also were revealed. Null alleles were evidenced at each calcium‐sensitive gene locus. At the CSN3 gene locus, we observed a prevalence of the CSN3 A and B alleles; the occurrence of rare alleles such as CSN3 B'', C, C', D, E and M; and the CSN3 S allele (GenBank KF644565 ) described here for the first time in Capra hircus. Statistical analysis showed that all genes, except CSN3, significantly influenced milk traits. The CSN1S1 BB and AB genotypes were associated with the highest percentages of protein (4.41 and 4.40 respectively) and fat (5.26 and 5.34 respectively) (P < 0.001). A relevant finding was that CSN2 and CSN1S2 genotypes affected milk protein content and yield. The polymorphism of the CSN2 gene affected milk protein percentage with the highest values recorded in the CSN2 AA goats (4.35, at P < 0.001). The CSN1S2 AC goats provided the highest fat (51.02 g/day) and protein (41.42 g/day) (P < 0.01) production. This information can be incorporated into selection schemes for the Sarda breed goat.     

Associations between milk performance traits in Holstein cows and 16 candidate SNPs identified by arrayed primer extension (APEX) microarray

An oligonucleotide microarray-which allows for parallel genotyping of many SNPs in genes involved in cow milk protein biosynthesis-was used to identify which of the 16 candidate SNPs are associated with milk performance traits in Holstein cows. Four hundred cows were genotyped by the developed and validated microarray. Significant associations were found between four single SNPs, namely DGAT1 (acyloCoA:diacylglycerol acyltransferase), LTF (lactoferrin), CSN3 (kappa-casein), and GHR (growth hormone receptor) and with fat and protein yield and percentage. Many significant associations between combined genotypes (two SNPs) and milk performance traits were found. The associations between the combined genotypes DGAT1/LTF and DGAT1/LEPTIN analyzed traits are presented as examples. The microarray based on APEX (Arrayed Primer Extension) is a fast and reliable method for multiple SNP analysis of potential application in marker-assisted selection. After further development, the chip may prospectively be used for dairy cattle paternity analysis and evolutionary studies.     

Sequences associated with human iris pigmentation

To determine whether and how common polymorphisms are associated with natural distributions of iris colors, we surveyed 851 individuals of mainly European descent at 335 SNP loci in 13 pigmentation genes and 419 other SNPs distributed throughout the genome and known or thought to be informative for certain elements of population structure. We identified numerous SNPs, haplotypes, and diplotypes (diploid pairs of haplotypes) within the OCA2, MYO5A, TYRP1, AIM, DCT, and TYR genes and the CYP1A2-15q22-ter, CYP1B1-2p21, CYP2C8-10q23, CYP2C9-10q24, and MAOA-Xp11.4 regions as significantly associated with iris colors. Half of the associated SNPs were located on chromosome 15, which corresponds with results that others have previously obtained from linkage analysis. We identified 5 additional genes (ASIP, MC1R, POMC, and SILV) and one additional region (GSTT2-22q11.23) with haplotype and/or diplotypes, but not individual SNP alleles associated with iris colors. For most of the genes, multilocus gene-wise genotype sequences were more strongly associated with iris colors than were haplotypes or SNP alleles. Diplotypes for these genes explain 15% of iris color variation. Apart from representing the first comprehensive candidate gene study for variable iris pigmentation and constituting a first step toward developing a classification model for the inference of iris color from DNA, our results suggest that cryptic population structure might serve as a leverage tool for complex trait gene mapping if genomes are screened with the appropriate ancestry informative markers.     

Relative efficiency of the linkage disequilibrium mapping approach in detecting candidate genes for schizophrenia in different European populations

Detection of susceptibility genes in indirect association studies depends not only on the degree of linkage disequilibrium between the disease variant and the SNP marker but also on the difference in their allele frequencies. Little is known about how variations in these parameters may affect the power of indirect association studies among related populations. Toward this goal, we genotyped 40 SNPs at four loci in samples from three European populations, Galician, Greek, and Norwegian. We compared the relative efficiency of all pairs of SNPs in detecting each other in each one of the populations. Our results show that a low percentage of marker SNPs may detect association in some populations but be totally ineffective in others. Therefore, these differences have to be an additional factor to consider when a replication study fails to confirm initial associations, especially if the replication is focused on very few markers.     

An allele associated with a non-detectable amount of αs2 casein in goat milk

The goat CSN1S2 locus is characterized by the presence of three alleles, A, B and C, all associated with about 2.5 g/l of protein per allele. The SDS-PAGE analysis of 441 individual milk samples obtained from goats belonging to a population reared in Southern Italy showed that the milk produced by three goats did not apparently contain alpha s2-casein, whereas milk produced by 37 goats showed a less intense electrophoretic band of this casein fraction (about 50%). These results can be explained by hypothesizing the presence of another allele at this locus, CSN1S2o, associated with a 'null' content of alpha s2-casein. Southern blot, PCR and PCR-RFLP analyses of the DNA region containing the CSN1S2 gene of individuals producing milk with and without alpha s2-casein did not show differences between the two groups. As a consequence, goats producing milk without alpha s2-casein carry an apparently intact gene. The first results obtained by sequencing part of the CSN1S2o allele revealed a G-->A transition at nucleotide 80 of the 11th exon which creates a stop codon and could be responsible for the absence of the alpha s2-casein in goat milk. This mutation eliminates a NcoI restriction site. A test based on this polymorphism has been established in order to identify carriers of the CSN1S2o allele.     

Associations Between Milk Performance Traits in Holstein Cows and 16 Candidate SNPs Identified by Arrayed Primer Extension (APEX) Microarray

An oligonucleotide microarray—which allows for parallel genotyping of many SNPs in genes involved in cow milk protein biosynthesis—was used to identify which of the 16 candidate SNPs are associated with milk performance traits in Holstein cows. Four hundred cows were genotyped by the developed and validated microarray. Significant associations were found between four single SNPs, namely DGAT1 (acyloCoA:diacylglycerol acyltransferase), LTF (lactoferrin), CSN3 (kappa-casein), and GHR (growth hormone receptor) and with fat and protein yield and percentage. Many significant associations between combined genotypes (two SNPs) and milk performance traits were found. The associations between the combined genotypes DGAT1/LTF and DGAT1/LEPTIN analyzed traits are presented as examples.

The microarray based on APEX (Arrayed Primer Extension) is a fast and reliable method for multiple SNP analysis of potential application in marker-assisted selection. After further development, the chip may prospectively be used for dairy cattle paternity analysis and evolutionary studies.     

The influence of extracellular matrix and prolactin on global gene expression profiles of primary bovine mammary epithelial cells in vitro

An in vitro bovine mammosphere model was characterized for use in lactational biology studies using a functional genomics approach. Primary bovine mammary epithelial cells cultured on a basement membrane, Matrigel, formed three-dimensional alveoli-like structures or mammospheres. Gene expression profiling during mammosphere formation by high-density microarray analysis indicated that mammospheres underwent similar molecular and cellular processes to developing alveoli in the mammary gland. Gene expression profiles indicated that genes involved in milk protein and fat biosynthesis were expressed, however, lactose biosynthesis may have been compromised. Investigation of factors influencing mammosphere formation revealed that extracellular matrix (ECM) was responsible for the initiation of this process and that prolactin (Prl) was necessary for high levels of milk protein expression. CSN3 (encoding κ-casein) was the most highly expressed casein gene, followed by CSN1S1 (encoding αS1-casein) and CSN2 (encoding β-casein). Eighteen Prl-responsive genes were identified, including CSN1S1 , SOCS2 and CSN2, however, expression of CSN3 was not significantly increased by Prl and CSN1S2 was not expressed at detectable levels in mammospheres. A number of novel Prl responsive genes were identified, including ECM components and genes involved in differentiation and apoptosis. This mammosphere model is a useful model system for functional genomics studies of certain aspects of dairy cattle lactation.     

Comparing the diversity of the casein genes in the Asian mouflon and domestic sheep

We aimed to determine whether casein variants that are currently segregating in ovine populations existed before the domestication of sheep or, to the contrary, if their emergence is much more recent. To this end, we have retrieved whole-genome sequences from Iranian and domestic sheep from Africa, Europe, South and East Asia and West Asia. Population structure analysis based on 55,352,935 SNPs revealed a clear separation between Iranian mouflons and domestic sheep. Moreover, we also observed a strong genetic differentiation between Iranian mouflons sampled in geographic areas close to Tehran and Tabriz. Based on sequence data, hundreds of SNPs mapping to the casein α_S1 (CSN1S1, 248 SNPs), casein α_S2 (CSN1S2, 268 SNPs), casein ß (CSN2, 146 SNPs) and casein κ (CSN3, 112 SNPs) genes were identified. Approximately 25–63.02% of the casein variation was shared between Iranian mouflons and domestic sheep, and the four domestic sheep populations also shared 44.2–57.4% of the casein polymorphic sites. These findings suggest that an important fraction of the casein variation present in domestic sheep was already segregating in the mouflon prior to its domestication. Genomic studies performed in horses and dogs are consistent with this view, suggesting that much of the diversity that we currently detect in domestic animals comes from standing variation already segregating in their wild ancestors.     

Comparative informativeness for linkage of multiple SNPs and single microsatellites

Single nucleotide polymorphisms (SNPs), or biallelic markers, are popular in genetic linkage studies due to their abundance in the genome, stability, and ease of scoring. We determined the 'information ratio' (IR) of closely spaced SNPs in simulated nuclear families and affected sib pairs (ASPs). (The IR is the ratio of actual average maximum lod score to the maximum lod score attainable if the marker were fully informative.) The nuclear families included parental information, whereas the ASPs did not. We analyzed these SNPs in two ways: (1) using multipoint analysis, and (2) treating the SNPs as 'composite markers' (i.e., haplotypes, as assigned by GENEHUNTER). (3) We also calculated the IR of a single microsatellite marker with multiple alleles and compared with the IR from the SNPs. For each set of input conditions, we simulated 1000 nuclear families, of 2, 3, 4, or 5 children each, as well as 1000 ASPs. We generated SNP marker data for strings of k = 1, 2, 3, 5, 7, and 10 SNP loci, with no recombination (theta = 0) and no linkage disequilibrium among the SNPs. The MAF (minor allele frequency) was either 0.5 or 0.25, and allele frequencies were the same for all k loci in any analysis. We also generated marker data for one single-locus microsatellite marker, with m = 3, 4, 5, 6, 7, and 9 equally frequent alleles. In all simulations, the disease was fully penetrant dominant, and there was no recombination or linkage disequilibrium among markers or between marker and disease. When multipoint analysis was used, we found that 5-7 closely spaced SNPs were usually enough to yield an IR of approximately 100%, for nuclear families of any size. However, for the ASPs, even 7-10 SNPs yielded an IR of only 70-80%. A microsatellite with 9 equally frequent alleles yielded about the same IR (86-88%) as a string of 4-5 SNPs, in nuclear families. SNPs analyzed as 'composite markers' analyses performed worse, due to the inherent ambiguity of SNP haplotyping.     

Development of a MLST-biased SNP microarray for Candida albicans

We have developed a single nucleotide polymorphism (SNP) detection microarray for the human pathogenic yeast, Candida albicans, consisting of synthetic oligonucleotides bound to microscope slides. The array consists of multiple replicates of 79 SNPs, derived from 19 discrete loci located on all eight chromosomes. These loci include seven genes consisting of 57 SNPs that comprise a multi-locus sequence typing (MLST) consensus scheme for the species. The remaining 22 SNPs are from 12 additional loci located at intervals on the remaining chromosomes. In order to include highly informative polymorphisms from the MLST set on the array we performed a linkage analysis of major genotypes between the two pairs of MLST-linked genes. In addition, we performed a matched-set analysis for each SNP located within individual MLST loci. This analysis resulted in the reduction of informatively redundant mutations in the array for a large percentage of strains. We believe that a SNP array will be helpful in extending our knowledge of the epidemiology and genetics of C. albicans as a supplement to MLST typing.     

The reliability of haplotyping inference in nuclear families: misassignment rates for SNPs and microsatellites

Single nucleotide polymorphisms (SNPs) are widely used when investigators try to map complex disease genes. Although biallelic SNP markers are less informative than microsatellite markers, one can increase their information content by using haplotypes. However, assigning haplotypes (i.e., assigning phase) correctly can be problematic in the presence of SNP heterozygosity. For example, a doubly heterozygous individual, with genotype 12, 12, could have haplotypes 1-1/2-2 or 1-2/2-1 with equal probability; in the absence of additional information, there is no way to determine which haplotype is correct. Thus an algorithm that assigns haplotypes to such an individual will assign the wrong one 50% of the time. We have studied the frequency of haplotype misassignments, i.e., haplotypes that are misassigned solely because of inherent marker ambiguity (not because of errors in genotyping or calculation). We examined both SNPs and microsatellite markers. We used the computer programs GENEHUNTER and SIMWALK to assign the haplotypes. We simulated (a) families with 1-5 children, (b) haplotypes involving different numbers of marker loci (3, 5, 7 and 10 loci, all in linkage equilibrium), and (c) different allele frequencies. Misassignment rates are highest (a) in small families, (b) with many SNP loci, and (c) for loci with the greatest heterozygosity (i.e., where both alleles have frequency 0.5). For example, for triads (i.e., one-child families with both parents genotyped), misassignment rates for SNPs can reach almost 50%. Family sizes of 4-5 children are required in order to ensure a misassignment frequency of < or = 5% for ten-SNP haplotypes with allele frequencies of 0.25-0.5. For microsatellites, a family size of at least 2-3 children is necessary to keep haplotyping misassignments < or = 5%. Finally, we point out that it is misleading for a computer program to yield haplotype assignments without indicating that they may have been misassigned, and we discuss the implications of these misassignments for association and linkage analysis.     

Geographic distribution of haplotype diversity at the bovine casein locus

The genetic diversity of the casein locus in cattle was studied on the basis of haplotype analysis. Consideration of recently described genetic variants of the casein genes which to date have not been the subject of diversity studies, allowed the identification of new haplotypes. Genotyping of 30 cattle breeds from four continents revealed a geographically associated distribution of haplotypes, mainly defined by frequencies of alleles at CSN1S1 and CSN3. The genetic diversity within taurine breeds in Europe was found to decrease significantly from the south to the north and from the east to the west. Such geographic patterns of cattle genetic variation at the casein locus may be a result of the domestication process of modern cattle as well as geographically differentiated natural or artificial selection. The comparison of African Bos taurus and Bos indicus breeds allowed the identification of several Bos indicus specific haplotypes (CSN1S1*C-CSN2*A²-CSN3*A_I/CSN3*H) that are not found in pure taurine breeds. The occurrence of such haplotypes in southern European breeds also suggests that an introgression of indicine genes into taurine breeds could have contributed to the distribution of the genetic variation observed.     

An SNP in the goat CSN2 promoter region is associated with the absence of beta-casein in milk

So far, at least eight alleles in the goat CSN2 locus have been associated with the level of β -casein expression in milk. Alleles CSN2 ^A , CSN2 ^{A 1}, CSN2 ^B , CSN2 ^C , CSN2 ^D and CSN2 ^E have been associated with normal content (allele effects of about 5 g of β -casein per litre), whereas the CSN2 ⁰ and CSN2 ⁰¹ alleles have been associated with non-detectable levels of β -casein. Most of these alleles have been characterized genetically. Herein, we report the identification of a previously unreported SNP in the goat CSN2 promoter region ( AJ011018 :g.1311T>C), which is associated with the absence of β -casein in the milk. Furthermore, we developed a PCR-based method that allows detection of this mutation.