J. Dairy Sci. 2007. 90:3522-3529. doi:10.3168/jds.2006-679
© 2007 American Dairy Science Association ®
Molecular Characterization of Bovine CSN1S2*B and Extensive Distribution of Zebu-Specific Milk Protein Alleles in European Cattle
E. M. Ibeagha-Awemu*,
,
E.-M. Prinzenberg*,
O. C. Jann*,
G. Lühken*,
A. E. Ibeagha,
X. Zhao and
G. Erhardt*,1
* Institute of Animal Breeding and Genetics, Justus-Liebig-University, Ludwigstrasse 21b, D-35390 Giessen, Germany
Department of Animal Science, Macdonald Campus of McGill University, Ste-Anne-de-Bellevue, Quebec, H9X 3V9, Canada
1 Corresponding author: georg.erhardt{at}agrar.uni-giessen.de
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ABSTRACT
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The B allele of the bovine 
S2-casein gene (CSN1S2) was characterized at the molecular level and the distribution of zebu-specific milk protein alleles was determined in 26 cattle breeds originating from 3 continents. The CSN1S2*B allele is characterized by a C 
T transition affecting nucleotide 17 of exon 3, which leads to a change in the eighth amino acid of the mature protein, from Ser to Phe (i.e., TCC TTC). DNA-based methods were developed to identify carriers of CSN1S2*B and the other alleles (CSN1S2*A, C, and D) at the same locus. CSN1S2*B and other zebu-specific milk protein alleles and casein haplotypes are widely distributed in European cattle breeds, particularly those of southeastern origin. Alleles CSN1S2*B and CSN3*H are important in searching for zebu imprints in European cattle breeds. Diversity estimates at the milk protein loci were highest in the zebus followed by southeastern European taurines. Anatolian Black had the highest number of zebu alleles among European taurines. Common, group, and intergroup haplotypes occurred in the breeds and demonstrated relationships that concurred with developmental histories, genetic makeup, and, in particular, exposed the extent of zebu influence on southeastern European cattle.
Key Words: S2-casein • zebu-specific alleles • Bos • haplotypes
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INTRODUCTION
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The S2-CN gene (CSN1S2), a member of the CN gene family, codes for S2-CN (CSN1S2), which constitutes up to 10% of the bovine CN fraction and exists in 2 major forms and several minor components. The reference protein form, CSN1S2*A, is a single polypeptide of 207 AA. Its sequence was originally determined chemically (Brignon et al., 1977) and later by cDNA and genomic DNA sequencing (Stewart et al., 1987; Groenen et al., 1993). In addition to CSN1S2*A, 3 other protein variants (B, C, and D) are known. Variants B and C are specific to zebu and yaks, respectively (Grosclaude et al., 1976, 1978, 1982), whereas the D variant has been found at low frequencies in some European breeds and the African Namchi taurine breed (Grosclaude et al., 1979; Erhardt, 1993; Jann et al., 2004; Ibeagha-Awemu et al., 2005a). The original variant A is the most frequent in all breeds so far investigated and also almost fixed in most western breeds. The different forms of the gene and protein are caused by single nucleotide polymorphisms (SNP) that result in amino acid changes. Single nucleotide polymorphisms in the CSN1S2*C protein result in AA changes at positions 33 (Glu to Gly), 47 (Ala to Thr), and 130 (Thr to Ile) compared with the A variant (Mahé and Grosclaude, 1982). Although CSN1S2*D differs from CSN1S2*A by the splicing-out of exon 8 (AA residues 51 to 59) due to an SNP in the genomic DNA sequence, the distinguishing feature(s) of CSN1S2*B variant have not yet been determined. In a recent analysis of African zebu cattle, Ibeagha-Awemu et al. (2005b) recorded CSN1S2*B at frequencies of 3 to 20%. Considering that this variant is also prevalent in other breed groups; for example, the Podolic cattle of Italy (Chianese et al., 1988), detailed information about its nature and distribution are therefore necessary.
Similar to the observation of CSN1S2*B in the Italian Podolic breed, other zebu-specific milk protein alleles and CN haplotypes have been reported in southeastern European breeds (Prinzenberg et al., 1999; Jann et al., 2004). Because some milk protein variants, like CSN1S2*B and LAA*A, were believed to exist in zebu breeds only, they are often ignored in milk protein studies of European cattle (Formaggioni et al., 1999). The extent of their occurrence and distribution in European cattle breeds is therefore poorly understood.
Alleles that display a higher or exclusive presence in zebus while showing a relatively low presence or complete absence in the other Bos species are denoted as zebu-specific alleles. Differences in allelic distributions can be used to assess the ancestry of populations or to determine relationships between different gene pools; this approach is similar to the concept of population-associated alleles defined by Kumar et al. (2003). MacHugh et al. (1997) first applied this concept to cattle populations. Subsequent investigations have expanded the list, including milk protein alleles and haplotypes, mitochondrial DNA haplotypes, and microsatellite alleles (Loftus et al., 1999; Mahé et al., 1999; Moazami-Goudarzi et al., 2001; Troy et al., 2001; Ceriotti et al., 2004; Cymbron et al., 2005; Ibeagha-Awemu et al., 2005a) and confirmed their usefulness in the study of genetic relationships between the Bos species. This approach thus contributes supportive molecular evidence, in addition to information from sex chromosomal analysis (Hanotte et al., 2000), necessary to substantiate historical and archeological claims of earlier and more recent contacts between zebu and European taurine cattle after their initial separation. Zebu-specific attributes may be more widely distributed in European cattle than suggested by current data.
The aims of the study were to characterize the CSN1S2*B protein variant at the molecular level and to determine its distribution and that of other zebu milk protein alleles in Bos taurus breeds.
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MATERIALS AND METHODS
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Sampled Breeds and Genes
A total of 1,185 unrelated individuals bearing typical breed characteristics of 26 breeds from 10 countries spanning 3 continents were sampled for the study (Table 1
). The breed groups include Indian zebu, African zebu, African taurine, and European taurine. Nelore and Brahman were recently imported into Brazil and Paraguay from India and are here considered as representations of Indian zebu genes. DNA was isolated from the blood of sampled animals according to the method of Montgomery and Sise (1990). Samples carrying the CSN1S2*B variant were selected for characterization at the DNA level. Specific zebu alleles at other milk protein loci [ S1-CN promoter (CSN1S1Prom), 
-CN (CSN3), and -LA (LAA)] were also genotyped. The S1-CN (CSN1S1) and ß-CN (CSN2) genes were also included.
Sequence Characterization of CSN1S2*B
Several primer pairs were designed to amplify the open reading frame of the cattle CSN1S2 gene (Gen-Bank No. M94327; Groenen et al., 1993), including exons 2 to 16 (Table 2). These sections in 3 individuals of the Banyo Gudali breed (2 homozygous and 1 heterozygous for the B allele; Ibeagha-Awemu et al., 2005b) were PCR amplified and analyzed for polymorphisms by sequencing. The PCR amplifications with their respective primers (Table 2) were carried out in a 20-µL reaction volume containing 50 ng of genomic DNA, 1 x PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3), 1.5 to 2.5 mM MgCl2 (Table 2), 200 µM dNTP, 10 pmol of each primer, and 0.5 to 2 U of Taq DNA polymerase (Eppendorf AG, Hamburg, Germany). The cycling conditions using the BioRad thermal iCycler (BioRad Laboratories GmbH, Mü nchen, Germany) included an initial denaturation at 94 ° C for 2 min followed by 30 cycles each of 94 ° C for 30 s, 53.7 to 62.5 ° C (Table 2) for 1 min, 72 ° C for 40 to 75 s (depending on size of fragment), and a final extension step at 72 ° C for 5 min. Before sequencing, the PCR products were purified using the Wizard SV Gel and PCR Clean-Up-System (Promega GmbH, Mannheim, Germany). Both sides of purified PCR products were sequenced using the big dye termination technique and an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA) following the manufacturer’s instructions. The sequences were processed with Chromas Version 1.45 (http://www.technel-ysium.com.au/chromas.html) and compared with the sequence of the bovine CSN1S2*A gene (GenBank No. M94327) with GeneDoc (Nicholas and Nikolas, 1997; http://www.psc.edu/biomed/genedoc).
Genotyping of Alleles at the CSN1S2 Locus (A, B, C, and D)
Based on the sequence characteristics of the alleles, methods employing restriction enzymes were developed to identify their presence. For CSN1S1*B, a 253-bp region encompassing an SNP characterizing the B variant was PCR amplified using the primers B S2_6090F and B S2_6342R (Table 2
). The 15-µL PCR reaction contained 50 ng of genomic DNA, 1 x PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3, and 1.5 mM MgCl2), 200 µM dNTP, 10 pmol of each primer, and 0.35 U of Taq DNA polymerase (Eppendorf AG). The PCR was performed in an iCycler (BioRad Laboratories GmbH) with the following conditions: initial denaturation for 2 min at 94 ° C followed by 30 cycles each of 30 s at 94 ° C (denaturation), 30 s at 59.3 ° C (annealing), and 1 min at 72 ° C (elongation), and a final extension for 10 min at 72 ° C. Amplified products were digested with 1 unit of MboII enzyme for 16 h at 37 ° C.
For alleles C and D, primers were designed to contain the respective characteristic mutations in exon 6 (Glu33 Gly33; i.e., GAG GGG) and 8 (G8879 T8879, last nucleotide of exon) of GenBank No. M94327. A 459-bp region was PCR amplified with the primers B S2_7360F and B S2_7818R for the C allele and a 356-bp region was amplified with the primers B S2_8675F and B S2_9030R for the D allele (Table 2). In both cases, the PCR reaction was in a final volume of 15 µL containing 50 ng of DNA, 1 x PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3, and 1.5 mM MgCl2), 200 µM dNTP, 10 pmol of each primer, and 0.35 U of Taq DNA polymerase (Eppendorf AG). The thermal profile (iCycler) included an initial denaturation for 2 min at 94 ° C followed by 30 cycles each of denaturation for 30 s at 94 ° C, annealing for 30 s at 53.7 or 60.5 ° C (Table 2), and elongation for 30 s at 72 ° C, and a final elongation for 10 min at 72 ° C. The resulting products were digested with 1 unit of NlaIV enzyme (allele C) or MnlI enzyme (allele D) for 16 h at 37 ° C. The resulting fragments were separated in 1% agarose gels and visualized by fluorescent absorption under UV radiation. The presence of the C allele was investigated in at least 20 random individuals of each breed homozygous for the A allele to exclude its occurrence in the studied breeds. Yak and German Red individuals heterozygous, respectively, for the C and D protein variants were used as standards (Erhardt, 1993).
Genotyping of Alleles at Other Milk Protein Loci
The methods of PCR-single strand conformation polymorphism (Prinzenberg et al., 2003) and PCR-RFLP (Mitra et al., 1998) were used to genotype alleles at CSN1S1Prom (alleles 1 to 5) and LAA (alleles A and B), respectively. Genes CSN1S1, CSN2, and CSN3 were genotyped by isoelectric focusing and PCR-single strand conformation polymorphism (Erhardt, 1993; Barroso et al., 1999; Prinzenberg et al., 1999).
Statistical Analysis
Allele frequencies at the loci studied were estimated with the GENEPOP program (Raymond and Rousset, 2001) and gene diversities (or expected unbiased heterozygosities) with the POPGENE program (version 1.31; Yeh et al., 1999). Haplotypes at the 4 CN loci, including the promoter region of CSN1S1 were determined with the program PHASE V2.1.1 (Stephens et al., 2001; Stephens and Donnelly, 2003). The PHASE program implements a Bayesian method of haplotype reconstruction based on genealogies reconstructed from coalescent theory under a Markov chain Monte Carlo framework and has been shown to outperform other strategies in most cases (Stephens et al., 2001).
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RESULTS
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Comparison of the sequenced regions of CSN1S2*B with CSN1S2*A (GenBank No. M94327) showed a single C T transition at nucleotide 17 in exon 3. This SNP changes the eighth AA of the mature protein, Ser8- Phe8 (TCC TTC) and also creates a restriction site for MboII restriction endonucleases, enabling identification of CSN1S2*B by RFLP. The MboII enzyme cuts the amplified 253-bp region, including exon 3, into 2 fragments of 126 and 127 bp for the B allele, whereas all other alleles remain uncut (data not shown).
Previously described mutations within the D and C variants also affected restriction sites and enabled their detection by restriction enzymes. For the CSN1S2*D allele, the recognition site of MnlI was disrupted by the SNP, resulting in an uncut 356-bp amplified product, whereas CSN1S2*A and other alleles were cut into 2 fragments of 160 and 196 bp. The exon 6 mutation in CSN1S2*C created a restriction site for NlaIV, which cut the amplified 459-bp region into fragments (211 and 248 bp) whereas other alleles remained uncut.
The distribution of these CSN1S2 alleles and alleles at the CSN1S1Prom, CSN3, and LAA loci in the breeds studied is shown in Table 3 and indicates the presence of zebu-specific milk protein alleles in European breeds. Frequencies of CSN1S2*B and LAA*A were higher in Nelore and Brahman than the African zebus and were also found with low frequencies in 6 European taurine breeds and 1 African taurine breed. The combined frequencies of alleles A1 and H of CSN3 were greatest in Brahman and Nelore, followed by the African zebus. Within the taurines, CSN3*A1 was only detected in Anatolian Black and Namchi, whereas CSN3*H was more widely distributed, with frequencies as high as 0.353 in Anatolian Black and 0.344 in the Turkish Gray Steppe. Zebu-specific allele 5 of CSN1S1Prom was more frequent in the African zebus than in the Indian zebu representatives; Chianina was the only European breed with this allele.
Genotypes at CSN1S1 and CSN2 were included in gene diversity analysis because one allele each at these loci occurred at frequencies above 50% in zebu breeds. Gene diversity estimates were higher in the zebus, southeastern European breeds, and the Cameroonian Namchi (taurine) than in other breed groups (Table 1).
Using genotypes of the 22 alleles detected at these loci; the PHASE program determined a total of 252 CN haplotypes (CSN1S1Prom-CSN1S1-CSN1S2-CSN2-CSN3; data not shown). Only those haplotypes that occurred at frequencies > 0.01 will be further reported. Our analysis showed haplotypes that were common to all breeds, specific to particular breed groups, or shared between certain groups (Figure 1), and further explains zebu influence on European cattle. Group-specific haplotypes were either completely absent or occurred at frequencies < 0.01 in the other groups. The haplotype 2BAA2B (CSN1S1Prom*2-CSN1S1*B-CSN1S2*A-CSN2*A2- CSN3*B) was common to all breeds, 1BAA2B to the taurines, and 1CAA2A1, 2CAA2A1, 2CBA2A, 2CBA2A1, and 5CAA2H to zebus. Other haplotypes separated the African zebus from the Indian zebus and the taurines into 3 groups (African, central European, and southeastern European taurines). Also, several haplotypes occurred across breed groups, in particular between zebus and southeastern European taurines and African taurines. Interestingly, 5 haplotypes (1CAA3H, 2CBA3H, 3BAA2H, 4BAA2H, and 5CBA2B) specific to southeastern European taurines contain the zebu-specific alleles CSN3*H or CSN1S2*B.
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Figure 1. Common, group-specific, and intergroup haplotypes explain breed relationships, especially zebu influence on southeastern European cattle. Haplotypes were estimated with the program Phase V2.1.1 and only haplotypes with frequencies > 0.01 are represented. 2BAA2B = CSN1S1PROM*2-CSN1S1*B-CSN1S2*A-CSN2*A2- CSN3*B; C1 to C10 represent intergroup haplotypes (C1 = 1BAA1B and 2CAA1B; C2 = 1CAA2H and 2CBA2H; C3 = 2BAA2H; C4 = 2CAA2H and 2CAA2H; C5 = 2BAA1A and 3BAA1B; C6 = 1CAA2B; C7 = 2BAA1B and 2BAA2A; C8 = 1CAA2A; C9 = 2BBA2H; C10 = 2CAA2B). Zebu-specific alleles that occur within southeastern European and African taurine haplotypes are highlighted; intergroup haplotypes that contain zebu-specific alleles are also highlighted.
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DISCUSSION
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We report here the SNP and AA change that distinguishes the B allele from other alleles at the CSN1S2 locus and also methods to identify carriers of this and other alleles at the locus. This study therefore provides necessary information that has been lacking about this locus (Farrell et al., 2004). Availability of this information will also facilitate the detection of this allele by DNA methods in individuals irrespective of age, sex, and physiological status compared with protein methods that make use of milk samples only. The identified SNP probably has an indirect effect on the physicochemical properties of the protein variant, because it involves the conversion of the first phosphorylated serine residue into a hydrophobic amino acid (phenylalanine). This hypothesis was not addressed in this study, however. The B variant was first reported in South African zebu breeds and in the yaks (Grosclaude et al., 1976, 1978) and is thought to be limited to these groups. In a study by Mahé et al. (1999) including 6 African B. taurus and indicus breeds, no polymorphism was identified at the protein level. However, Chianese et al. (1988) found this protein variant in the Italian Podolian cattle with a very low frequency but no further effort was made to characterize the AA substitutions and to include this variant in the characterization of other European breeds. Our study, besides having characterized the CSN1S2*B allele, shows its wider presence, as well as of CSN3*H in European breeds, mainly of southeastern origin like Chianina (Italian Podolian breed), Piemontese, Anatolian Black, and Turkish Gray Steppe, and Polish Red of central European origin. Frequencies of CSN1S2*B as well as other zebu-specific milk protein alleles (CSN3*A1, CSN3*H, and LAA*A) are highest for Nelore and Brahman, intermediate in African zebus, and lowest in all taurine breeds and are clearly related to the geographic origin of the breed, which confirms similar observations made by Beja-Pereira et al. (2002) and Jann et al. (2004). CSN1S1Prom*5 is the zebu-specific allele that seems to have a wider distribution in African zebu than in Indian zebu breeds, which may indicate a role in adaptation to hotter climates or simply be the direct result of genetic drift.
With the exclusion of Polish Red, European breeds lacking the zebu-specific milk protein alleles are all from central Europe and are also highly specialized breeds. This fact consequently affected estimates of milk protein genetic diversities for these breeds. The low presence of these alleles in the central European breeds could be attributed to their zebu origin, or some forces of selection could have played a role. In a recent study, Jann et al. (2004) reported a significant decrease of genetic diversity in European cattle breeds from the south to the north and from the east to the west and attributed this to the process of domestication as well as natural and artificial selection. Also, high selection pressure for the milk and meat traits of central European cattle breeds may have contributed to the absence of these alleles, considering that all cattle once originated from a common ancestor. When we talk of a common ancestor, the haplotype that was common to all the breeds investigated is further proof of this. Also, all alleles within the common haplotype (2BAA2B) identified in this study are considered to be the wild-type allele at their respective loci except for CSN3*B (Prinzenberg et al., 2003; Farrell et al., 2004; Ibeagha-Awemu et al., 2005a).
Characterization of CSN1S2*B and genotyping of alleles at this locus in more Bos taurus and Bos indicus cattle breeds has enabled a better definition of group-specific CN haplotypes compared with Mahé et al. (1999). This study has identified haplotypes that occur exclusively in the various breed groups. Additionally, some intergroup haplotypes and southeastern European haplotypes containing zebu-specific alleles showed the extent of zebu gene flow into these breeds. It is evident from our work that the occurrence of more haplotypes than reported by Jann et al. (2004) and Ibeagha-Awemu et al. (2005a) is caused by the occurrence of CSN1S2*B in African cattle and in some European cattle.
The haplotype information has further elucidated the extent of existing relationships among the breeds, particularly the zebu influence on southeastern European cattle and the Polish Red, and also the genetic subdivisions within European cattle breeds. Breed groups and intergroup haplotypes are further clues about the genetic makeup and historical development of the breeds (Mason, 1996; MacHugh et al., 1997; Troy et al., 2001; Ibeagha-Awemu et al., 2004).
Adding to our findings, Beja-Pereira et al. (2006) observed that different southern European cattle breeds were affected by introgression of breeds from northern Africa and that the previous simple hypothesis regarding the domestication process should be revised. In particular, the greater genetic diversity of the southeastern European breeds is the consequence of the presence of alleles of zebu origin and, therefore, indicates extensive hybridization with zebu cattle. Geographic proximity and early historical associations between the Mediterranean region and North Africa may explain the zebu genetic influence in southern European cattle. For example, Cymbron et al. (2005) showed that breeds in the Mediterranean region (Italy, Greece, and Portugal) had a greater frequency of zebu-associated microsatellite alleles (6.7%) than did breeds in the rest of Europe (5.1%). A T1 mitochondrial DNA haplotype characteristic of African breeds (Bradley et al., 1996; Troy et al., 2001) was shown by Anderung et al. (2005) to be present not only in prehistoric (1,800 yr ago) Iberian cattle but also in present-day populations, thus giving evidence of prehistoric and recent contact between African pastoralists and the Iberian Peninsula. This input from zebu cattle has also lead to increased genetic diversity of southeastern European cattle breeds and might indicate an extensive hybridization zone (Loftus et al., 1999; Jann et al., 2004; Cymbron et al., 2005) rather than reduced CN diversity of more specialized breeds by selection.
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ACKNOWLEDGEMENTS
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This work was supported by the German Academic Exchange Service (DAAD) and in part by the European Commission (RESGEN-CT98-118). The content of this publication does not represent the views of the Commission or its services. The authors thank Christel Zörb for excellent technical support.
Received for publication October 17, 2006.
Accepted for publication February 14, 2007.
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