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J. Dairy Sci. 88:1878-1881
© American Dairy Science Association, 2005.

Short Communication: Predominance of ß-Casein (CSN2) C Allele in Goat Breeds Reared in Italy

S. Chessa1, E. Budelli2, F. Chiatti1, A. M. Cito3, P. Bolla1 and A. Caroli3

1 Dipartimento di Scienze e Tecnologie Veterinarie per la Sicurezza Alimentare, Università degli Studi di Milano, 20134 Milano, Italy
2 Fondazione Parco Tecnologico Padano, Centro Ricerche e Studi Agroalimentari, 20090 Segrate (Milano), Italy
3 Dipartimento di Sanità e Benessere Animale, Università di Bari, 70010 Valenzano (Bari), Italy

Corresponding author: Anna Caroli; e-mail: am.caroli{at}veterinaria.uniba.it.


   ABSTRACT
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 ABSTRACT
ACKNOWLEDGEMENTS
 REFERENCES
 
A protocol for the rapid and simultaneous genotyping of A, C, and 0 'CSN2 alleles in goat was developed by single strand conformational polymorphism polymerase chain reaction (SSCP-PCR) technique. Screening the CSN2 variability in 7 goat breeds reared in Italy validated the genotyping test. The SSCP-PCR technique was also suitable for monitoring CSN2 polymorphism. In particular, the discrimination between CSN2*A and CSN2*C is important because the 2 corresponding protein variants cannot be separated by standard typing techniques. The monitoring of CSN2 variability in the goat breeds indicates the predominance of the C allele. In most breeds, CSN2*C occurred with the highest frequency, except in Saanen where CSN2*A and CSN2*C showed similar frequencies. Variant CSN2*C occurred with a frequency of 0.68 (Camosciata), 0.70 (Jonica), 0.71 (Garganica), 0.82 (Maltese), 0.87 (Cilentana), and 0.97 (Orobica). The alignment among the mature CSN2 sequences of different species suggests that CSN2*A is the ancestral allele compared with CSN2*C. Interestingly, the CSN2*A goat variant showed higher frequencies in selected breeds (Saanen and Camosciata).

Key Words: goat • ß-casein • genetic polymorphism • single strand conformational polymorphism

Abbreviation key: CSN2 = ß-casein, IEF = isoelectrofocusing, SSCP = single strand conformational polymorphism

Genetic polymorphism of milk proteins has been intensively studied in goat because of the deep relationships with functional and biological properties affecting milk quality, composition, and technological characteristics (Martin 1993; Grosclaude et al., 1994). Goat caseins show a complex qualitative and quantitative variability, characterized by several genetic polymorphisms as well as by multiple post-translation modifications. Different transcriptional and post-transcriptional mechanisms control casein gene expression, dramatically affecting the technological properties of milk (Martin et al., 2002).

Casein genes are organized as a cluster including in the order {alpha}s1-casein, ß-casein (CSN2), {alpha}s2-casein, and {kappa}-casein loci (Ferretti et al., 1990; Threadgill and Womack, 1990). Within the cluster, the first 2 casein loci are only 12 kb apart and convergently transcribed (Leroux and Martin, 1996). In goats, the entire casein gene cluster region spans about 250 kb on chromosome 6 (Hayes et al., 1993; Popescu et al., 1996).

As far as CSN2 is concerned, 3 protein variants were found to be associated with a normal ß-casein content: A, B, and C. The B variant was detected by isoelectrofocusing (IEF), resulting in some bands markedly closer to the gel cathodic position than in the A variant (Mahé and Grosclaude, 1993). Genetic control of the B allele was supported by the segregation analysis in one available family. However, the variant was not further characterized. The C variant was identified by peptide mass fingerprinting and tandem mass spectrophotometry (Neveu et al., 2002). This variant differs from the A in the mono amino acid substitution Ala177 to Val177 of the mature protein. Because both amino acids are neutral, the mutation is not detectable by screening protein techniques such as milk IEF. At the DNA level, the protein polymorphism is justified by a nucleotide substitution GCA (Ala177) -> GTA (Val177), as can be observed by alignment of the sequence GenBank accession number AF409096 (Wang et al., 2001; direct submission) with AH001195 sequence (Roberts et al., 1992).

Furthermore, 2 different null CSN2 alleles were identified, both characterized by mutations responsible for premature stop codons in exon 7, one in southern Italian breeds (Ramunno et al., 1995; GenBank accession number AJ011019) and the other in the Creole and Pyrenean goat (Persuy et al., 1999; GenBank accession number AF172260). The mRNA analysis revealed that the transcript product amounts were almost 10 (Ramunno et al., 1995) and 100 (Persuy et al., 1999) times lower for the null alleles than for the A variant. As far as the nomenclature is concerned, the 2 null alleles were differentiated respectively by 0 (AF172260) and 0' (AJ172260) by Neveu et al. (2002). This nomenclature will be used in the present communication.

To obtain further information on goat CSN2 variability, exon 7 was analyzed by single strand conformational polymorphism- (SSCP-) PCR in 7 Italian goat breeds. An SSCP-PCR protocol was developed for typing CSN2*0' as an alternative to allele-specific PCR (Ramunno et al., 1995). Different SSCP patterns were detected and sequenced. Moreover, milk samples were typed by IEF to check the correspondence between the DNA variation and the phenotypic expression.

Blood and individual milk samples were randomly collected from 4 Southern Italian goat breeds (Garganica, Jonica, Cilentana, and Maltese), 1 Northern Italian breed (Orobica), and 2 composite breeds (Camosciata and Saanen). Four hundred seventy-three samples were analyzed. Reference samples for the CSN2*0' allele (GenBank accession number AJ011019) were also used. A commercial kit (GFXTM Genomic Blood DNA Purification kit, Amersham Biosciences, Piscataway, NJ) was used for DNA extraction from blood or milk.

A 374-bp fragment containing exon 7 of the goat CSN2 gene was amplified by PCR performed in a 25- µL reaction mixture containing 2 µL of DNA solution (100 to 150 ng), 10 pmol of each primer, and 1xPCR Master Mix (Fermentas, Vilnius, Lithuania). Primers were 5'CCC AAA GTG AAG GAG ACT ATG 3', and 5' CAT CAG AAG TTA AAC AGC ACA G 3'. The following conditions were used: an initial denaturation step of 95°C for 2 min, followed by 30 cycles of 94°C for 45 s, 58°C for 45 s, 72°C for 2 min, and a final extension step of 72°C for 5 min using a PTC-0200 DNA Engine thermal cycler (MJ Research Inc., Waltham, MA).

For SSCP, 6 µL of PCR product was added to 8 µL of denaturation solution (0.05% of xylene-cyanol, 0.05% of bromophenol blue, 0.02 M EDTA in deionized formamide). After heat denaturation at 95°C for 8 min, the samples were immediately chilled on ice and then run overnight (16 h) on 10% acrylamide:bisacrylamide gels (29:1) with 1.5% glycerol in 0.5x Tris-borate-EDTA buffer at 280 V and 12°C (Penguin Dual Gel Water-Cooled Electrophoresis System, OWL Scientific Inc., Woburn, MA). Bands were visualized by silver staining (Bassam et al., 1991).

The DNA samples showing different patterns on SSCP gels were randomly selected for sequencing. Eight samples were sequenced. Primers used for sequencing were the same as those used for the PCR-SSCP techniques. The PCR products were sequenced by PRIMM Srl, Milano, Italy. The nucleotide sequences and the deduced amino acid sequences were analyzed with Bioedit (Hall, 1999) software.

Milk samples were typed for CSN2 by IEF according to Caroli et al. (2001).

The use of the PCR-SSCP method developed gave the opportunity to identify the CSN2*C allele at the DNA level simultaneously with CSN2*A and CSN2*0' (Figure 1Go). Three main bands with increasing electrophoretic mobility were identified and attributed respectively to CSN2*C, A, and 0' on the basis of sequencing results or reference sample comparison, or both. The correspondence between the CSN2*C protein variant and SSCP was demonstrated by sequencing (GenBank AY563136). The nucleotide exchange C-> T was found in all the sequenced samples presenting the CSN2*C band (2 AC, 1 C0', 2 CC), whereas it did not occur in the other 3 DNA samples sequenced (2 AA, 1 A0').



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  		Figure 1. Single strand conformational polymorphism-PCR typing for the simultaneous detection of CSN2*A, CSN2*C, and CSN2*0' (GenBank Accession number AJ011019). Genotypes are indicated for each sample. The discriminant band for each allele is also shown (star: CSN2*C, circle: CSN2*A; triangle: CSN2*0').

 
Thus, the PCR-SSCP technique is particularly suitable for the study of CSN2 variability. The discrimination between the A and C variants is important because the 2 variants are not identifiable at the protein level by standard typing methods, as shown by IEF. This technique allows separating 2 major and 2 minor bands of CSN2, simultaneously with alleles at other casein loci (Caroli et al., 2001; Erhardt et al., 2002). The occurrence of the B variant was excluded by comparing our results with the literature (Mahé and Grosclaude, 1993). Moreover, the comigration of A and C variants at the protein level was confirmed by IEF, and the presence of new phenotypes in the typed breeds was excluded.

The monitoring of CSN2 variability in goat breeds reared in Italy clearly indicates the predominance of CSN2*C. Except for Saanen, where CSN2*A and CSN2*C showed similar frequencies, CSN2*C occurred with a higher frequency than CSN2*A. Frequencies of CSN2*C were 0.68 (Camosciata), 0.70 (Jonica), 0.71 (Garganica), 0.82 (Maltese), 0.87 (Cilentana), and 0.97 (Orobica) (Table 1Go). The CSN2*0' allele was found only in the Italian southern breeds, at frequencies ranging from 0.046 (Garganica) to 0.093 (Cilentana and Jonica). No individual homozygous for CSN2*0' allele was observed, as expected on the basis of Hardy-Weinberg equilibrium, which was verified in all breeds. No search was performed for the null allele CSN2*0 identified in Creole and Pyrenean goat (Persuy et al., 1999), and, in the future, it would be useful to include this variant in the PCR-SSCP typing test for goat CSN2.


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  		Table 1. Number of individuals typed (n) and allele frequencies at CSN2 locus in the different breeds. Frequencies are shown in ascending order for CSN2*C.
 
From a phylogenetic point of view, the alignment among the mature CSN2 sequences of different species (Figure 2Go) suggests that CSN2*A is the ancestral allele compared with CSN2*C. In the figure, protein sequences (http://www.ebi.ac.uk/swissprot/) are shown starting from the amino acid position 158 of caprine CSN2. Sequences 1 and 2 are deduced, respectively, from goat CSN2*A (Roberts et al., 1992) and CSN2*C (Wang et al. 2001, direct submission) alleles. In the other ruminants (sequence 3 = sheep; 4 = bovine; 5 = buffalo), alanine occurs in the position corresponding to goat amino acid 177, thus suggesting a more ancestral origin of goat CSN2*A if compared with CSN2*C. However, valine occurs at the 177th correspondent position in sequences 6 (pig), 7 (human), and 8 (rabbit), which, as expected, exhibit a higher divergence from caprine CSN2 than ruminants. In all cases, the fact that CSN2*A goat variant shows higher frequencies in selected breeds (Saanen and Camosciata) than in isolated populations should be further investigated in the light of functional and adaptive aspects, and taking into account the genetic polymorphism at the other casein genes.



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  		Figure 2. Alignment of the mature CSN2 of different species, starting from the amino acid position 158 of the caprine CSN2, to which the headline refers. Sequences 1 and 2 are deduced, respectively, from CSN2*A (Roberts et al., 1992) and CSN2*C (Wang et al., 2001; direct submission) goat variants. Web site: http://www.ebi.ac.uk/swissprot/.

 
Comparison with literature data on goat CSN2 allele frequencies is difficult due to the low level of polymorphism detected at this locus until now. No indication is given about the incidence of the C allele in the different breeds by the authors who identified this variant (Neveu et al., 2002). The 0' allele was found in a local southern Italian population with a frequency of 0.1 (Rando et al., 1996). The 0 allele was found in a large flock of Creole goats (Mahé and Grosclaude, 1993) and in thousands of animals of the Pyrenean breed (Persuy et al., 1999) with a frequency of 0.2 and 0.12, respectively.

In conclusion, CSN2*C is the predominant allele in goat breeds reared in Italy. The SSCP-PCR test developed is a rapid method to screen goat breeds at the CSN2 locus. The demonstrated polymorphism should be considered in addition to the already known variants at the other casein loci, to include casein haplotype information in breeding programs for the genetic improvement of domestic goats.


   ACKNOWLEDGEMENTS
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 ABSTRACT
 ACKNOWLEDGEMENTS
REFERENCES
 
The DNA reference samples carrying the CSN2*0' allele were a kind gift from Luigi Ramunno and coworkers. The research was partially supported by MURST contract year 2002 – prot. 2002072131.

Received for publication October 19, 2004. Accepted for publication December 20, 2004.


   REFERENCES
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 ABSTRACT
 ACKNOWLEDGEMENTS
 REFERENCES
 


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Hayes, H., E. Petit, C. Bouniol, and P. Popescu. 1993. Localisation of the alpha-S2-casein gene (CASAS2) to the homologous cattle, sheep and goat chromosome 4 by in situ hybridisation. Cytogenet. Cell Genet. 64:282–285.

Leroux, C., and P. Martin. 1996. The caprine alphaS1- and beta-caseins genes are 12 kb apart and convergently transcribed. Anim. Genet. 27(Suppl. 2):293.

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