Polymorphism of the kappa-casein gene in two Bosnian autochthonous cattle breeds

Buša is an old endangered autochthonous breed of the western Balkan, especially Bosnia-Herzegovina, Kosovo and Albania. A related breed is Gatačko, derived from Buša × Tirolean Grey crossbreds. Fifteen purebred Buša cattle and thirteen Gatačko animals were genotyped for polymorphisms at the kappa-casein gene by a Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) essay. The alleles A, B and C were found and the allelic frequencies were 0.46 (A), 0.46 (B) and 0.08 (C) in Buša cattle and 0.58 (A) and 0.42 (B) in Gatačko. Only AA, AB, BB and BC genotypes occurred. Further alleles were not detected and are therefore either absent in both populations or rare. The allele »B« found in this small population will be useful for a sire selection program in the future.


Introduction
The Buša breed is an old autochthonous cattle-breed of the western Balkan, namely Bosnia and Herzegovina (BiH), Croatia, Serbia, Kosovo and Albania.While in former days there were thousands of Buša cows in BiH, population size has sharply decreased and nowadays the population counts less than 100 heads.Small size (roughly 102-112 cm), low body weight (200-250 kg) and an average milk yield of about 2 000 kg per lactation are among the characteristic features of the breed.Due to their low body weight Buša animals are thought to fit especially well in low-input production systems.
The Gatačko breed has been derived from Buša × Tirolean Greys hundred years ago, and is only found in BiH.Mean body weight (250-300 kg) and milk yield (2 500 kg) are somewhat higher than in the Buša breed.The current population size is estimated as about 200 heads.For more information on both breeds the reader is referred to ADILOVIĆ et al. (2005); Figure 1 gives a visual impression on the visual impression of Buša and Gatačko animals.The genetic polymorphisms presented by milk proteins are transmitted by simple Mendelian inheritance with no dominance.The casein gene has a role to stabilise the casein micelles and by this it also stabilizes the production characteristics of milk which is particularly relevant in cheese production.So far, six varieties of this gene have been described: A, B, C, E, and, recently, also F and G varieties (KAMINISKY 1996).Many studies show the influence of the genetic variety of kappa-casein on the production characteristics of milk.Milk from animals which have the B variety of the gene shows better lactodinamographic characteristics than milk from animals with the A variety (RAHALI and MÉNARD 1991), E variety (GRAVERT et al. 1991) or C variety (MACHEBOEUF et al. 1990) The purpose of this work was to investigate for the first time the occurrence and frequency of CSN3-alleles from tissue samples of the BiH autochthonous cattle breeds Buša and Gatačko in order to get insight into an important aspect of both the genetic variability of these severely endangered cattle breeds and the cheese-making properties of their milk.

Material and methods
In total 28 full-blood samples were collected, 15 of them from BiH autochthonous Buša cattle, which were kept on several farms in middle and north-western Bosnia.Additionally 13 samples of full blood were drawn from BiH Gatačko cattle from a single farm in Gacko.For isolation of genomic DNA standard methods were used (MITRA et al. 1998).

Amplification of the information segment of the kappa-casein gene
After the previously described analyses, amplification of a fragment of 453 BP by using the standard PCR (Polymerase Chain Reaction) method with the following parameters: F 5'-TGT GCT GAG TAG GTA TCC TAG TTA TGG-3' and R 5'-GCG TTG TCT TCT TTG ATG TCT CCT TAG-3' (BARROSO et al. 1998) was performed.457 l PCR supermix (Cat# 10572-014, Invitrogen) containing (22 mM Tris-HCl, 55 mM KCl, 1.65 mM MgCl2, 220 μM each of dNTP, 22/L recombinant Taq polymerase stabilisator), per 20 pmol from both primers and 1/L DNA extract was used in this reaction.Size and yield of PCR product is characterised by the application of agarosis gel-electrophoresis on 1 % agarosis gel (MANIATIS et al. 1982) (Biorad Laboratories, Cat#162-0126), and the findings were documented as described below.The reactions followed the sequence: one cycle at 95 °C for 30 s (initial denaturation), and 30 cycles of the sequence: 95 °C for 60 sec, 58 °C for 60 s and 72 °C for 45 s and 7 min for final elongation at 72 °C.After the reaction was completed, fragments were subjected to electrophoresis in an (1.5 %) agarose gel, at 90 V for approximately 1.5 h.Visualization of the bands was done under ultraviolet Trans-illumination and a picture was taken with KODAK Edas system for gel documentation.The size of the amplified product was compared against 50 bp Ladder DNA marker (Fermentas) for qualitative analysis.

Genotyping of kappa-casein gene by PCR-RFLP method
In the process of genotyping of the amplified gene segment containing the information sites for kappa-casein polymorphism enzymes Hinf I (Invitrogen) -with specific spot -5'-GANTC-3' , Hae III (Invitrogen) -5'-GGCC-3' and HpyCH4 (New England Biolabs) -5'-ACGT-3' have been used.HpyCH4 is isoschizomer for Mae II, an enzyme used in a similar study (BAROSSO et al. 1998).Digestion conditions for all used enzymes were as follows: 1X of appropriate restricting buffer, 5U enzyme, 15 μL PCR product and dH2O 20 μl of the total volume.Three separate reactions are incubated at 37 °C for the duration of 16 h.Products of restriction analyses are detected on the AGOROZNOM gel dyed by etidiumbromid according to the protocol from MANIATIS et al. (1982).Nomination and interpretation of genotypes were according to BAROSSO et al. (1998).

Results and discussion
Documented results of agarose-electrophoresis of genomic DNA show that the samples are of sufficiently good quality and concentration of minimally 20-50 ng/l, which is a sufficient quantity for the amplification to be performed on such a matrix (Figure 2 and 3).The sample analysed by MOODY et al. (1996) contained alleles A, B and C, allele B being the most frequent.The CSN3 allele and genotype frequencies considerably varied in different cattle breeds studied (Table ).The frequencies of allele B varied from 0.42 to 0.46; and those of genotypes AB and BB, from 0.35 to 0.53 and from 0.15 to 0.21, respectively.The frequency of the B-allele was higher in Buša-cows compared to Gatačko-cattle.In other breeds studied, the frequency of the B allele is high and varied from 0.25 to 0.32; and that of the BB genotype, from 0.03 to 0.09.The BB genotype frequency was extremely high in both breeds and in comparison to other breeds the frequency of the B-allele was considerably high.
Frequencies of both major alleles were not far from 0.5 in both populations.It seems remarkable that both breeds showed considerable genetic variation despite of their small effective population size.The considerably high frequency of the B-allele provides a genetic foundation for good cheese-making properties of the milk from the autochthonous breeds.The frequency of the B-allele may even be further increased by selection, however in doing so the alleles at closely linked milk protein loci should also be regarded (e.g.CZERNIAWSKA-PIĄTKOWSKA) as well as polymorphisms of other genes with possible impact on milk quality (e.g.DYBUS et al. 2004 andkappa-casein effects on other traits (e.g. DYBUS et al. 2005).The test used for the determination of the genetic variants of genes for kappa-casein based on the PCR-RFLP method allows both rapid and efficient examination of the variations of this gene, regardless of the age of animals.In this way it is possible to establish and increase the frequency of desired alleles among animals in studied populations included in the programs of selection and preservation of the autochthonous animal genetic resources in Bosnia and Herzegovina.

Figure 2
Figure 2 Restriction fragment pattern of PCR-products from the kappa-casein gene Restriktionsmuster der PCR-Produkte des Kappa-Kasein Gens

Figure 3
Figure 3 Genotyping results using Tai I (fragments with lengths of 453, 254 and 199 bp are visible) Ergebnisse der Typisierung mit Tai I (mit sichtbaren Fragmenten der Längen 453, 254 und 199 bp) . MITRA et al. (1998), used for the first time the PCR-RFLP technique with restriction enzymes Hind III, Hinf I and Taq I, this technique made it possible to detect alleles A and B of the kappa-casein gene (CSN3) in Sahiwal cattle (Bos indicus), and in Murrah, Nili-Ravi and Egyptian buffaloes (Bubalus bubalis).A primary PCR product of 379 bp length was amplyfied in a first step.The enzymes Hind III and Taq I produced two fragments of allele B: 156 and 223 bp, and 123 and 256 bp, respectively.Digestion with Hinf I resulted in three fragments of 132, 156 and 91 bp, respectively, for allele A of gene CSN3, and two fragments of 288 and 91 bp, respectively, for allele B of gene CSN3, with a frequency of 0.16 in (Bos taurus) cattle.In the Sahiwal breed, 39 animals were identified with genotype CSN3 AA, and the other 18 with genotype CSN3 AB.The genotype BB, however, was not detected among the animals studied.DOGRU and OZDEMIR (2009) observed three genotypes in Brown Swiss with frequencies 19.35, 20.43 and 60.22 % for AA, BB and AB.

Table Distribution of
genotypes in the sample Verteilung der Genotypen in der Stichprobe