Short communication: predominance of beta-casein (CSN2) C allele in goat breeds reared in Italy.

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).

Casein haplotype structure in five Italian goat breeds.

The aim of this work was to investigate the genetic structure of the casein gene cluster in 5 Italian goat breeds and to evaluate the haplotype variability within and among populations. A total of 430 goats from Vallesana, Roccaverano, Jonica, Garganica, and Maltese breeds were genotyped at alphas1-casein (CSN1S1), alphas2-casein, (CSN1S2), beta-casein (CSN2), and kappa-casein (CSN3) loci using several genomic techniques and milk protein analysis. Casein haplotype frequencies were estimated for each breed. Principal component analysis was carried out to highlight the relationship among breeds. Allele and haplotype distributions indicated considerable differences among breeds. The haplotype CSN1S1*F- CSN1S2*F-CSN3*D occurred in all breeds with frequencies >0.100 and was the most common haplotype in the Southern breeds. A high frequency of CSN1S1*0-CSN1S2*C-CSN3*A haplotype was found in Vallesana population (0.162). Principal component analysis clearly separated the Northern and Southern breeds by the first component. The variability of the caprine casein loci and variety of resulting haplotypes should be exploited in the future using specific breeding programs aiming to preserve biodiversity and to select goat genetic lines for specific protein production.

Effects of casein haplotypes on milk production traits in Italian Holstein and Brown Swiss cattle.

The objective of this study was to estimate the effects of different haplotypes of the casein genes on milk production traits in Italian dairy cattle. Traits of interest were yields of milk, fat, and protein, and percentages of fat and protein in milk. The data included 728 multiparous records from 347 Holsteins and 773 records from 298 Brown Swiss cows. Records were preadjusted for effects of age and parity, season of calving, and region, and expressed as deviations from herdmate averages. Twenty half-sib families were represented in each breed. Haplotype probabilities were estimated for each animal and phenotypes were regressed on these probabilities. Nine haplotypes were observed in Holsteins and 17 were identified among the Brown Swiss. For Holsteins, significant effects were observed for protein percentage, with some indication of an effect for fat percentage. For the Brown Swiss, effects of haplotypes were significant for milk yield and fat and protein percentages. Effects were strongest for protein percentage. Correlation coefficients of solutions across breeds tended to be strong and positive, indicating that the same haplotypes had similar estimated effects in the 2 breeds. Although the data were limited (<350 cows in each study), this latter result may suggest that genes in the casein complex itself are responsible for the effects observed, rather than loci that are physically linked on either side of the casein cluster.

Effect of udder health status and lactation phase on the characteristics of Sardinian ewe milk.

Mammary involution and inflammation are known to negatively affect milk quality. A trial was carried out to elucidate the mechanism by which udder health status and lactational phase determine compositional modifications in ovine milk. A total of 60 individual milk samples was collected from a group of 20 pluriparous Sardinian ewes from mid to late lactation. Each sample was assessed for its chemical characteristics, quantitative distribution of casein fractions, lactodynamographic characteristics, and enzymatic activity. Udders were classed as healthy, doubtful, or infected on the basis of repeated somatic cell counts, and samples were grouped in 3 classes of days in milk. Results indicated that both udder inflammation and mammary involution can increase plasmin (PL) activity (15.6 vs. 18.4 U/mL in healthy vs. infected udders; 14.0 vs. 20.2 U/mL in phase 1 vs. 3), which is responsible for an evident protein breakdown in milk. Significant differences between groups were observed for several characteristics. With regard to udder heath status, casein index was lower in the infected vs. healthy udders (74.8 vs. 76.6%), and beta(tot)-casein showed a similar trend (43.9 vs. 46.6%). As a consequence of protein degradation, gamma-casein (5.78 vs. 2.82%) and proteolysis index (7.60 vs. 3.82) increased in the infected group with respect to the healthy group. Udder health status also affected milk technological traits. Udder inflammation resulted in longer clotting time (20.7 vs. 16.5 min for infected vs. healthy, respectively) and in poorer curd firmness (35.6 vs. 47.6 mm for infected vs. healthy, respectively). Frequency of samples reactive to rennet was 100, 93, and 67%, respectively, for healthy, doubtful, and infected groups. With regard to lactational phase, a decrease in alpha(s1)-casein (39.13 vs. 29.36%) and beta(1)-casein (23.41 vs. 19.36%) occurred during phase 1 vs. 3, whereas kappa + alpha(s2)-casein increased (12.30 vs. 21.56%, phase 1 vs. 3). Correlation coefficients confirmed the role of PL in protein degradation. It was concluded that PL activity was strongly affected by both lactational phase and udder health status and, in turn, could be an important agent enhancing milk quality detriment.

Single nucleotide polymorphisms in the ovine casein genes detected by polymerase chain reaction-single strand conformation polymorphism.

Casein genetic polymorphisms are important and well known due to their effects on quantitative traits and technological properties of milk. At the DNA level, polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) allows for the simultaneous typing of several alleles at casein loci, as well as the detection of unknown polymorphisms. Here we describe the usefulness of the PCR-SSCP technique for casein typing in sheep. In particular, three single-nucleotide polymorphisms (SNP) are described at CSN1S1, CSN2, and CSN3, all resulting in amino acid exchanges. At CSN1S1, a transition T-->C was found, resulting in the deduced amino acid exchange Ile186-->Thr186. A transition A-->G resulting in the deduced amino acid exchange Met183-->Val183 was identified at CSN2. The 2 SNP showed a rather high frequency (ranging from 0.12 to 0.26) in 3 Italian breeds (Sarda, Comisana, Sopravissana). Another transition C-->T (Ser104-->Leu104) was found at CSN3 in one heterozygous animal.

Short communication: Simultaneous identification of five kappa-casein (CSN3) alleles in domestic goat by polymerase chain reaction-single strand conformation polymorphism.

Until now, a total of nine polymorphic sites corresponding to six different alleles have been described at the kappa-casein (CSN3) locus in the domestic goat (Capra hircus). A protocol for the rapid and simultaneous genotyping of five goat CSN3 alleles by using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique was developed. Moreover, the developed test was validated by screening the CSN3 variability in four Italian breeds, Garganica, Jonica, Maltese, and Camosciata. Seven different patterns were readily identifiable. These corresponded to five known alleles and two newly identified variants. The G/A substitution at nucleotide position 471, which is not identifiable at the protein level but was found to be very frequent in the typed breeds, is easily detectable by the protocol developed. The PCR-SSCP analysis is a powerful tool for the genetic study of CSN3 variability in domestic goats, allowing both the simultaneous identification of different alleles, and the detection of new variants.

Genetic polymorphism of goat kappa-casein (CSN3) in different breeds and characterization at DNA level.

Data on genetic differences at CSN3 in goat breeds including a DNA based typing method and the mutations responsible for variation on protein and DNA level are presented here. Isoelectric focusing (IEF) in ultrathin polyacrylamide gels with carrier ampholytes was used to demonstrate CSN3 polymorphism in milk samples of Italian (Orobica n=88; Ionica n=68) and German goat breeds (Bunte Deutsche Edelziege n=244; Weisse Deutsche Edelziege n=134; Toggenburger n=25; Thüringer Waldziege n=70). A further CSN3 band was found presenting a more cathodic migration than CSN A. After chymosin action, the genetic polymorphism was also observed in the para-kappa-casein fraction. The new allele CSN3(B) was spread mainly in Orobica (37%), Bunte Deutsche Edelziege (11%) and Ionica (10%). CSN3(B) occurred in low frequency (<3%) in Thüringer Waldziege and in Weisse Deutsche Edelziege, and could not be demonstrated in milk samples of Toggenburger. The populations were in Hardy-Weinberg equilibrium at CSN3. The codominant genetic control of CSN3(B) was confirmed by genetic studies. Discrimination of CSN3 alleles A and B was also obtained by DNA SSCP analysis. Sequencing of CSN3(B) revealed four transitions at position 247, 309, 471, and 591 compared with CSN3(A). From these transitions, the following amino acid substitutions are deduced: 44 Gln --> Arg, 65 Val --> Ile, 119 Val --> Ile, and 159 Ser --> Pro. Among the four mutations, only the 44 Gln --> Arg can be revealed by milk protein IEF analysis while at DNA level three further genetic variants should exist in addition to CSN3(A) and CSN3(B).

Single nucleotide polymorphisms (SNPs) within Bov-A2 SINE in the second intron of bovine and buffalo k-casein (CSN3) gene.

The genetic polymorphism of an entire Bov-A2 element located in the second intron of the buffalo and bovine k-casein (CSN3) gene was investigated by amplification and sequencing of PCR products. Single nucleotide polymorphisms were detected. A PCR-RFLP method was developed to detect an A or G mutation at position 605 of bovine Bov-A2 element which creates a BfaI polymorphic site. The frequencies of the B allele, with the BfaI site, were for 0.275, 0.775, 0.750, 0.975, respectively, for Italian Holstein Friesian, Grey Alpine, Friuli Red Pied and Reggio bovine breeds. The mutation rate (substitutions and deletions/insertions per nucleotide site per year) was 2.5 x 10(-9) for Bov-A2 sequences in the second intron of CSN3. The comparison with other Bov-A2 elements suggests that this retroelement might be an important source of single nucleotide polymorphism for analysis of Bovidae genomes.