1 Genetic structure and conservation of the Grey Steppe breed in RomaniaŞteofil Creangă, Elena Ruginosu, Lucian Dan Dascălu, Ionuţ Borş Research-Development Station for Bovine Growing - S.C.D.C.B. Dancu - Iaşi INTRODUCTION The Grey Steppe breed populations from Romania (fig. 1) have registered in the last 30 – 40 years an obvious decline, so in these days, the breed is threatened by extinction. That is why the preservation of this breed is highly required. Grey Steppe breed is the only breed of cattle ancestral Romanian, actually being encountered in small flocks in Iasi, Neamt, Delta, being conserved in a compact nucleus of 60 heads at SCDCB Dancu Iaşi. This race is important for its role in history, the genetic characters of strength and adaptability, unique, imposing its preservation as endangered breed. On the point of view of the analysis of genetic structures for lactoproteins genes localization, revealed this race has a distinct genetic structure.Also at αS1 casein locus identified a new allele, αS1 - IRV, which demonstrates oldness of that race, the identification of this allele is first in the podolitic family (figure 2). This new casein can be an important genetic marker of the breed. Genetic analysis of the Grey Steppe breed can provide many surprises in terms of understanding the mechanism of disease resistance and optimization of feeding a low nutritional value, improved breeds characters are reduced or lost. MATERIALS AND METHODS Fig. 1 Grey Steppe Breed, S.C.D.C.B. Dancu Iaşi (original photo) Researches were effectuated on 30 Romanian Grey Steppe cows raised semi-intensively, tied-up stalling, at the Research-Development Station for Bovine Growing Dancu, Iaşi (S.C.D.C.B. Dancu, Iaşi). Due to the strictly genetic determinism of milk proteins, what makes genotype be identical to phenotype, milk protein frequency is very different from one breed to another. Hence, the need to run these researches that might establish the genotypic and allelic frequencies of milk proteins for Romanian Grey Steppe breed, the Moldavian variety from the North-Eastern part of Romania. The study of polymorphism of milk proteins was made by PCR-RFLP technique, and for the study of polymorphism of all bovine milk proteins we also used the isoelectric focalization technique (I.E.F.) The milk samples were collected individually in 15 ml Falcon tubes, transported at 40 C and then frozen at -200C until tests were run. Defrosting occurred slowly at room temperature and subsequently, samples were centrifuged at rotations/minute, for 5 minutes for milk separation. They were stored for 30 minutes at 4 degrees for fat solidification and then it was removed from each tube by means of a spatula (Bâlteanu et al., 2007, 2008, Creangă, 2010). For an optimal protein concentration, samples were diluted with a urea and β-mercaptoethanol solution. Samples were migrated in a polyacrilamide gel with 4% concentration. After migration, the gel was immersed in a solution 10% of trichloroacetic acid. Coloring occurred for 2 hours by means of a solution 0.025% Coomassie Brilliant Blue R-250 in 40% ethanol and 7% glacial acetic acid. To estimate the correlations between milk quality indicatorshas been used the R.E.M.L method. (Restricted Maximum Likelihood). This method is based on an iterative process of maximizing a function. Computing techniques vary depending on the chosen optimization algorithm, but all of them require, at each iteration cycle, BLUP solutions for different effects of the model. A large number of iterations is needed to reach convergence, but this is unavoidable if an effectiv evaluation is desired. It is also a mixed model because it includes a random factor (animal) and two fixed factors (farm and rank lactation). RESULTS Milk has a complex chemical composition, especially in terms of casein fractions, whey proteins, enzymes and antimicrobial substances. Mutations that occurred over time in the structure of genes encoding the six major milk proteins have led to several genetic variants in these loci. These variations, named polymorphisms, indicates that milk protein species may be present in two or more autosomal genes coded forms (alleles) which have co-dominance interaction, which means that both alleles are expressed in heterozygous individuals. Knowledge of these genetic polymorphisms has great practical application in both selection and genetic improvement of animals and the conservation of animal genetic resources. Analysis of electrophoretic profiles revealed at the 6 loci coding for the six major types of milk protein, the presence of common alleles, but also of rare alleles, difficult to identify by PCR. Genetic structure for the polymorph systems of milk proteins: alpha-S1 casein (αS1-cz), beta-casein (β-cz), kappa-casein (K-cz), beta-lactoglobulin (β-lg), alpha-lactoalbumina (α-LA) and alpha-casein S2 (αS2-cz) is presented in Table 1 (Creangă et al., 2008, 2009, 2010, Vlaic et al., 2011, Jann et al., 2004, Ceriotti et al., 2004). As it is mentioned in the specialized literature (Thompson et al., 1962) in our case also s1-Cn B is more common, with a higher frequency of 0,7 and s1-Cn C allele at the studied animals had a 0,2 frequency. Casein s2 is monomorphous for Romanian Grey Steppe nucleus under study, for allele s2-Cn A, as it appears in all bovine breeds studied so far. Casein  has the two universal variants -Cn A1 and -Cn A2 found out at bovines and. In our case, allele -Cn A2 (0.550) is the most frequently met and -Cn A1 has a frequency of 0.45, variants B, C and A3 being absent. Variant -Cn A1 has a higher frequency for the breeds originating in North-West Europe and the breeds of improved members of the Bovidae family. The higher frequency of allele A2 has a special significance since this allele is the ancestral one from which all the others derived. All researches have undoubtedly showed the favorable influence of variant k-Cn B on milk quality, cheese output and quality. Consequently, in the study of bovine lactoproteins, most researches focused on the determination of the frequency of kappa-casein alleles at different breeds and the possibility of “limited” promotion by selection of kappa casein B. Variants k-Cn A and k-Cn B are universally discovered at bovines and zebu. In recent years, 3 more variants have been identified: k-Cn C, k-Cn D and k-Cn E, all having frequencies lower than 0.1 and being identified only in some local breeds. TABLE I - Genetic polymorphism of milk proteins for romanian grey steppe breed ISM genetic variant sequence and those of B and C, have been deducted based on mutations observed after sequencing of cDNA's and protein sequencing. To summarize, the ISM variant is characterized by the presence at position 99 (position 84 in mature protein) of amino acid Asp (D), version B showing in this position a Glu (E); in position 207 (position 192 in mature protein) variant ISM is characterized the presence of a Gly (G), version B showing in this position a Glu (E). The ISM variant differs from C variant by the presence of aminoacid Asp (D), which replaced th Glu (E) in position 99 (84 in the mature protein). In position 207 (192 in the mature protein). The genetic variants C and ISM are identical (fig. 4).Calculation of the isoelectric points of the majore proteins αS1-CN B, ISM and C (4,62; 4,67; 4,68) explains those mobility differences previously observed in IEF. According to those differences, the ISM variant migrate closer to the C instead of B (Bâlteanu et al., 2007; 2008, Creangă et al., 2010), conform cărora varianta ISM (fig. 4).Comparing the final results obtained at protein and DNA level, it can be concluded that the Glu-Gly substitution from position 207 (192 of mature protein), which differentiates the B from C and ISM allele, was confirmed by sequencing both protein and DNA. The other mutation that characterizes the ISM allele, could not be highlighted by Maldi Tof-MS analysis. It was identified by sequencing cDNA and located in the 11 exon. Registration no. α S1-cz β-cz K-cz β-lg α-la α S2-cz 9991 BIRV A1A2 AB BB AA 9993 A1A1 9983 9988 0004 CIRV A2A2 9985 BC 9990 9998 9723 9986 Genotype frequency BB = 0.5 BC = 0.3 CIRV = 0.1 BIRV = 0.1 A1A1 = 0.2 A1A2 = 0.5 A2A2 = 0.3 AA = 0.209 AB = 0.41 BB = 0.375 AA = 0.292 AB = 0.50 BB = 0.208 BB = 1 AA = 1 Allele frequency pB = 0.7 qC = 0.2 r IRV = 0.1 pA1 = 0.45 qA2 = 0.55 pA1 = 0.417 qA2 = 0.583 pA1 = 0.542 qA2 = 0.458 PB = 1 pA = 1 . 2 IEF profile for bovine milk proteins of Romanian Grey Steppe (lanes 1, 4, 5, 6, 8), as against Bălţata Românească breed (lanes 2, 3, 7) for locus of S1 casein. Genotypes of S1 casein are:1- BB; 2- BC; 3- CC; 4- CIRV; 5- BIRV; 6- BB; 7- BC; 8- BB. Phylogenetic origin of the ISM allele was derived from molecular data available. Mutation that occurred in exon 11, involving A-T substitution in position 21 (responsible for the Glu to Asp substitution in position 84 of mature protein), make the difference between ISM allele compared with alleles B and C. These data together with the difference between B and ISM allele in position 31 of the exon 17 (responsable for Gly – Glu substitution in position 192 of mature protein) and the similarity between C and ISM alleles in this position, suggest ISM allele has phylogenetically derived directly from the C allele (fig. 5). By putting together all the existent data available in the specialized literature and the data resulted in this study studiu, it can be concluded that the ISM alle is the only allele known to date in αS1-CN locus, at Bos taurus, derived directely from the ancestral allele C and not B (fig.5). A similar mutational event led to the aparition of allele Eyak (Bos gruniens), which also have its origin in allele C (Caroli et al, 2004, Formaggioni et al., 1999). Therefore, a new allele of αS1-CN, called ISM, discovered at the Grey Steppe breed, Moldavian variety, was fully characterized. It is a genetic marker that provides evidence about the phylogenetic origin of this breed and its ancestral progenitor. Using a combined methodology of protein and DNA sequencing, we were able to identify mutations that characterize this new allele of αS1-CN, thus confirming the assumption of this new genetic variant. To determine the relationship between the major milk protein systems, we investigated the correlation coefficients through phenotypic, genetic and environmental correlations (table 2). Fig. 4 Comparison between protein sequences features of B, C and IRV variants (Romanian Grey Steppe breed). The mutations, which are making the difference between this 3 protein variants, are marked with rectangles. Note the differences between isoelectric points (pI) of the 3 proteins, which explain the observed IEF profiles (gel image from the right side). Signal peptide is highlighted with bold italic letters. Table. 2 - Phenotypic correlations, genetic and environmental systems between the main protein in milk, at the Grey Steppe breed Fig. 5 Phylogenetic origin of alpha S1 IRV allele Character 1 Character 2 Phenotypic correlation Genetic correlation Environment correlation Genetic covariance Interlot covariance Total covariance S1- cz K-cz -0.21 0.27 -0.25 0.132 -0.3 -0.168 -cz 0.56 0.48 0.65 0.7 0.568 _lg -0.26 0.25 -0.28 0.18 -0.12 0.37 0.43 0.39 0.36 0.2 0.16 -lg 0.45 0.127 0.5 0.373 Analyzing phenotypic and environmental correlation coefficient values between S1 casein (S1-cz) and other three protein systems ( K-cz, β-cz, β-lg) results that for the K-casein (K-cz) β lactoglobulin (β-lg) the correlations are negative and the intensity is moderate. There is a signifiant antagonism between the two properties, and the β casein (β-cz) correlations are positive and with high values ​​(0.48 to 0.65%), indicating the same causes and determinants of the two groups of properties. Genetic correlations for all variants analyzed are within the positive and significant correlations. It may be noted that for these protein systems, the selection for milk protein systems can be done simultaneously thanks to the positive correlations of these traits. These are favorable for achieving improve milk quality for Grey Steppe breed. CONCLUSIONS The results we obtained show that the Grey Steppe breed individuals from Dancu farm, Romania, have a valuable genetic background to be preserved and improved for both milk and meat production. Also have to be developed in terms of numbers, in order to avoid the genetic drift and the close inbreeding. Molecular data have shown that αS1-CN ISM is the only genetic variant described to date in Bos taurus, having its phylogenetic origin in the ancestral variant αS1-CN C ((the most common genetic variant in primitive breeds from Asia and Africa), as is the αS1-CN E described at Yak (Bos grunniens). Another interesting observation is that αS1-CN ISM has never been observed in other cattle breeds in Europe, nor other breeds of cattle belonging to the Grey Steppe and not even at Hungarian Grey Steppe, Transylvanian variety (Baranyi et al., 1993) or other cattle breeds from Romania. The presence of this new αS1-CN allele was observed only at individuals of pure breed from SCDB Dancu farm, Iasi county, demonstrating this way that the origin of the αS1-CN ISM allele is indisputable. References Bâlteanu, A. V., Pop, D. F., Vlaic, A., Carsai, C. T., Creangă, Şt., Rusu, R. A – Characterization of the s1 Casein IRV allele provides evidence for phylogeny of the ancient Romanian Grey Steppe cattle, Moldavian strain. Lucrări ştiinţifice seria zootehnie CD, vol 53 (15) U.Ş.A.M.V. 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