The aim of this study was to identify polymorphisms in the bovine genes encoding CSN1A1 and CSN3 caseins and analyse the structure of population of Slovak Spotted cattle. Subsequent analysis was carried out to estimate the effect of both polymorphisms on long-life milk production traits. The genomic DNA was extracted from totally 180 blood samples. All of analysed animals were genotyped by using the PCR-RFLP method. In population the prevalence of homozygote animals was found. For CSN1A1 gene the BB genotype was noted as predominant. In terms of CSN3 gene the genotype frequencies was more balanced. The most frequent genotype were AA. The population genetic indices signalised the decrease of genetic variability in population mainly for CSN1A1 gene. Except FIS index, each of parameter reflected the high proportion of BB homozygous individuals in population. The values of observed and expected heterozygosity for CSN3 gene indicated similarly the prevalence of homozygote individuals in population, but the decrease of heterozygosity was not so high. The effect of polymorphisms on production traits was tested based on the GLM procedure. In case of both SNPs we were able to describe the variability of analysed traits on 95%. The study results clearly indicated the positive effects of CSN1A1BB and CSN3AA genotypes in order to improve the milk production traits in Slovak Spotted cattle. In addition, the statistical analysis confirmed the key role of casein in milk production and its composition

Anna Trakovická

Nina Moravčiková

Radovan Kasarda

English

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255Agriculturae Conspectus Scientifi cus . Vol. 82 (2017) No. 3 (255-258)ORIGINAL SCIENTIFIC PAPERSummaryTh e aim of this study was to identify polymorphisms in the bovine genes encoding CSN1A1 and CSN3 caseins and analyse the structure of population of Slovak Spotted cattle. Subsequent analysis was carried out to estimate the eff ect of both polymorphisms on long-life milk production traits. Th e genomic DNA was extracted from totally 180 blood samples. All of analysed animals were genotyped by using the PCR-RFLP method. In population the prevalence of homozygote animals was found. For CSN1A1 gene the BB genotype was noted as predominant. In terms of CSN3 gene the genotype frequencies was more balanced. Th e most frequent genotype were AA. Th e population genetic indices signalised the decrease of genetic variability in population mainly for CSN1A1 gene. Except FIS index, each of parameter refl ected the high proportion of BB homozygous individuals in population. Th e values of observed and expected heterozygosity for CSN3 gene indicated similarly the prevalence of homozygote individuals in population, but the decrease of heterozygosity was not so high. Th e eff ect of polymorphisms on production traits was tested based on the GLM procedure. In case of both SNPs we were able to describe the variability of analysed traits on 95%. Th e study results clearly indicated the positive eff ects of CSN1A1BB and CSN3AA genotypes in order to improve the milk production traits in Slovak Spotted cattle. In addition, the statistical analysis confi rmed the key role of casein in milk production and its composition.Key wordscasein, cattle, genetic polymorphism, milk yield and composition, PCR - RFLPCasein Polymorphism in Relation to the Milk Production Traits of Slovak Spotted Cattle Anna TRAKOVICKÁNina MORAVČIKOVÁ ( )Radovan KASARDA1 Department of Animal Genetics and Breeding Biology, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 94976 Nitra, Slovakia e-mail: nina.moravcikova@uniag.skReceived: May 1, 2017 | Accepted: August 19, 2017ACKNOWLEDGEMENTS This work has been supported by the Slovak Research and Development Agency (Contract No. APVV-14-0054 and No. APVV-0636-11).Agric. conspec. sci. Vol. 82 (2017) No. 3256 Anna TRAKOVICKÁ, Nina MORAVČIKOVÁ, Radovan KASARDAIntroductionMain goals of the dairy cattle breeding are the search of an economically effi  cient way of the improvement of milk produc-tion and the qualitative milk traits. Selection and breeding of animals with desirable genotypes is of crucial importance for the genetic improvement of dairy cows. Milk protein genetic polymorphisms of the genus Bos provoke a signifi cant scientifi c interest, mainly associated with their evolution, population struc-ture, breeding and hybridization. Over the last decades, studies have been concentrated on the infl uence of the genetic variants of the major milk proteins on the quantitative and qualitative milk traits and their technological properties (Hristov et al., 2012).Casein is a family of milk proteins that exists in several mo-lecular forms and is the main protein present in the bovine milk. Casein content in bovine milk ranges from 2.4% to 2.6%, which represents about 78% of all milk proteins (Żukiewicz et al., 2012). Bovine milk proteins are classifi ed by two groups; one group is soluble at pH 4.6, on the other hand, the other group is not sol-uble at pH 4.6. Th e soluble fraction is called whey protein such as α lactalbumin (LALBA), β lactogloulin (LGB). However, the insoluble part constitutes four casein protein such as α s1 casein (CSN1S1), α s2 casein (CSN1S2), β casein (CSN2) and κ-caseins (CSN3) (Gurcan, 2011; Gras et al., 2016). Caseins are encoded for by single autosomal genes that occupy approximately 200 Kbp on the sixth chromosome (Zwierzchowski et al., 2001). Th e linked genes are present in the sequence: αS1, β, αS2, κ, which means that their expression during lactation is regulated in a synchronic way. It is possible that a LCR (locus control region) exists for the casein genes (Żukiewicz et al., 2012). Most of the studies have been focused on CSN1S1 and CSN3 of the group of caseins and LGB of the whey proteins. Th ese proteins have a great eff ect on milk production and milk components. Th e ge-netic variants of the CSN3 gene have a dominant role due to the protein infl uence of this protein on the formation, structure and stabilization of the casein micelles with respect to milk techno-logical properties and cheese production (Hristov et al., 2012; Deb et al., 2014).Five genetic variants of CSN1S1 have been identifi ed: A, B, C, D, and E. Th e allele B is of the highest frequency in Bos taurus, whereas C is the most common in B. indicus and B. grunienns (Eigel et al., 1984). Th e allele B encodes glutamine whereas C encodes glycine, both at position 192 (Żukiewicz et al., 2012). Th e CSN3 gene comprises a 13 kb sequence divided into 5 exons (Martin et al., 2007). Th is gene encoded protein that aff ect the yield, composition as well as technical properties of milk. It is a standout amongst the most vital milk proteins in the mammals that assumes a crucial part in the milk quality and coagulation. Up to now, within the sequence of CSN3 gene eleven genetic variants have been distinguished (Trakovická et al., 2012). Th e most widely recognized alleles are A and B that diff er in two amino acids at position 136 (Th r → Ile) and 148 (Asp → Ala) (Awad et al., 2016).Th e aim of this study was to identify polymorphisms in CSN1S1 and CSN3 genes and to analyse their eff ect on long-life milk production traits in population of Slovak Spotted cattle.Material and methodsIn the study, overall 180 Slovak Spotted cow have been in-cluded to determine the impact of genes located in bovine casein locus on long-life milk production traits. Th e Slovak spotted cattle, belonging to the dual-purpose production type, is genetically re-lated with Simmental cattle and has a long tradition in Slovakia.Th e genomic DNA was extracted from blood samples ac-cording to protocol of Miller et al. (1988). Subsequently, the concentration and purity of genomic DNA were tested based on the spectrophotometry measurements by the optical den-sity at wave length of 260 nm. Th e polymorphisms in CSN1S1 and CSN3 genes were analysed according to Miluchová et al. (2014) using RFLP methods. Th e products of PCR reaction and restriction fragments have been separated and visualised using horizontal electrophoresis in 2% agarose gels (130 V for 50 min) and stained with day GelRed.Th e genotypic structure of population and allele frequen-cies have been determined using Genalex version 6.1 (Peakall and Smouse, 2012). Th e Hardy-Weinberg equilibrium in popu-lation was tested based on the signifi cance between observed and expected genotype frequencies using Chi-square test. Th e diversity indices including observed (Ho) and expected het-erozygosity (He), eff ective allele numbers (Ne), polymorphic information content (PIC), and FIS index was calculated using Genalex version 6.1 (Peakall and Smouse, 2012). Associations of the animal genotypes with long-life milk production traits were determined by analyses of quantitative traits. Statistical analysis was performed using SAS Enterprise Guide 4.2 soft ware (SAS Institute Inc., 2009) and signifi cance of diff erences based on genotypes eff ect of production traits were tested by follow-ing general linear models:Yijklm1,2,3 = Gi + BTj + Sk + Ll + BYm + eijklmwhere:  Yijklm1,2,3 – milk, protein and fat yield, Gi – eff ect of genotype, BTj – eff ect of breed type, Sk – eff ect of sire, , Ll – eff ect of number of lactations, BYm – date of birth, eijklm – random error.Results and discussionIn population of Slovak Spotted cows both analysed SNPs was successfully genotyped. For the CSN3 gene have been identifi ed all three genotype (AA, AB, and BB). In case of CSN1A1 only two genotype was detected (BB and BC), the CC genotype was not observed. Th e allele and genotype frequencies are listed in Table 1. For CSN1A1 gene the BB genotype was predominant in population, which also indicated considerably higher frequency of B allele in comparison to C allele. In terms of CSN3 gene the genotype frequencies was more balanced. Th e most frequent genotypes were AA and AB genotypes. Th e lowest frequency was found for homozygous BB genotype. In case of both SNPs the diff erences between observed and expected genotype fre-quencies were not signifi cant that indicate the Hardy-Weinberg equilibrium in population and low impact of eff ect such as selec-tion, inbreeding or migration. However, the population genetic indices signalised the decrease of genetic variability in popula-tion mainly for CSN1A1 gene (Table 1). Except FIS index, each of parameter refl ected the high proportion of BB homozygous Agric. conspec. sci. Vol. 82 (2017) No. 3257Casein Polymorphism in Relation to the Milk Production Traits of Slovak Spotted Cattleindividuals in population as well as the absence of CC genotype. Th e values of observed and expected heterozygosity for CSN3 gene indicated similarly the prevalence of homozygote individ-uals in population, but the decrease of heterozygosity is not so high. Th e FIS index on level 0.17 refl ects slightly increase of ho-mozygosity in population. According to the classifi cation of PIC (low polymorphism if PIC value < 0.25, median polymorphism if 0.25 < PIC value < 0.5, and high polymorphism if PIC value > 0.5) (Botstein et al., 1980), the analysis show low (CSN1A1) or median (CSN3) levels of polymorphic information content of SNPs across population.  Th e eff ectiveness of loci allele impact in populations has been expressed by eff ective allele numbers. Comparison of Ne showed higher eff ective allele numbers across populations for CSN3 gene and indicated good level of genetic variability in analysed population at the considered locus.Th e obtained allele and genotype frequencies of CSN1A1 and CSN3 genes are in agreement with previously published studies. Similarly to our results Kučerová et al. (2006) found in population of Czech Simmental cattle the predominance of CSN3A allele. In Czech Fleckvieh Bartoňová et al. (2012) confi rmed the higher proportion of CSN3A across analysed individuals. Keating et al. (2007) showed that this proportion of CSN3 allele frequencies is valid in various dairy cattle breeds. In the Slovakia, the CSN3 polymorphism was analysed in Simmental and Holstein cattle. Trakovická et al. (2012) reported in both breeds the predomi-nance of A allele over the B allele. Table 2 shows the observed average values of analysed pro-duction traits in population. Th e average values for each trait in relation to the specifi c CSN1A1 and CSN3 genotypes are listed in table 3. Th e statistical signifi cance of observed diff erences in association analyses was aff ected by the number of data available for particular trait. Th erefore, only animals with relevant data of production traits were used in subsequent analysis. In case of both SNPs we were able to describe the variability of analysed traits on 95%. Except genotypes eff ect, four other fi xed eff ects were included in the GLM model: sire, breed type, total number of lactation in long-life production, and date of birth. As expected due to the biology of milk production each of them showed high statistical signifi cant impact on milk, pro-tein as well as fat yield (Table 3).Th e statistical analysis confi rmed the key role of casein in milk production and its composition. In both SNPs higher milk production was found in the most frequent homozygous Locus Genotypes frequency Alleles frequency χ2 test Ho He Ne PIC FIS CSN1S1 BB BC CC B C ns 0.04 0.04 1.04 0.04 -0.019 0.96 0.04 - 0.98±0.01 0.02±0.01 CSN3 AA AB BB A B ns 0.35 0.42 1.74 0.34 0.17 0.51 0.36 0.13 0.69±0.02 0.31±0.02 Ho – observed heterozygosity, He – observed heterozygosity, Ne – effective allele number, PIC – polymorphic information content, FIS – fixation index,  ns – not significant Trait N Mean SD Min Max MY (kg) 180 19395.31 15372.86 733.00 55102.00 FY (kg) 180 702.06 544.50 23.00 2007.00 PY (kg) 180 619.17 495.91 22.00 1763.00 n – number of individuals, mean – average value, SD – standard deviation, Min – minimum, Max – maximum, MY – milk yield, FY – fat yield,  PY – protein yield Genotypes N Traits (in average) Factors MY (kg) FY (kg) PY (kg) CSN1S1 Breed type*** Sire*** No. of lactations** Birth date** BB 169 19719.67*** 713.0814*** 629.5349*** BC 8 12421.38*** 465.125*** 396.25*** CSN3 AA 91 22357.25** 812.02 716.02** AB 62 15481.63** 555.66 489.66** BB 24 18477.67** 670.96 593.25** MY – milk yield, FY – Fat yield, PY – protein yield, ** P<0.01, *** P<0.0001 Table 1. Summary of genotype structure and population indices evaluated in populationTable 2. Basic statistical variation measurements of long-life milk production in analysed populationTable 3. Impact of fi xed eff ects included in GLM model on milk production traits in analysed populationAgric. conspec. sci. Vol. 82 (2017) No. 3258 Anna TRAKOVICKÁ, Nina MORAVČIKOVÁ, Radovan KASARDAgenotypes in population. Cows of homozygous CSN1A1BB geno-type were characterized by highest milk, protein and fat yield (P < 0.0001). Th e opposite relationships between milk production and CSN1A1 genotypes were found in the group of cows with CSN1A1BC genotype. Th e statistical analysis of CSN3 genotype eff ect on long-life milk production traits shows signifi cant diff er-ences (P < 0.001) only for milk and protein yield. Th e genotype CSN3AA was favourable for each of analysed production traits.Th e signifi cant impact of caseins on milk production has been reported in many studies. In comparison to our results Micinski et al. (2007) reported the superiority of the BC geno-type in population, but they confi rm the positive impact of B allele on milk yield. Similarly Hristov et al. (2013) confi rmed the predominance of CSN1A1B allele over the CSN1A1C allele and its signifi cant impact mainly in regard to milk yield. For CSN3 genotypes Curi et al. (2005) reported the association between genotype AA and higher milk production. However, Strzalkowska et al. (2002) found that the BB genotype is favour-able for milk fat percentage. ConclusionTh e statistical analysis showed signifi cant eff ect of both an-alysed SNPs in genes encoding CSN1A1 and CSN3 caseins on long-life milk production of Slovak Spotted cattle. Th e study results indicated the positive eff ects mainly in relation to the CSN1A1BB and CSN3AA genotypes in order to improve the eco-nomically important traits for farmers. 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Casein Polymorphism in Relation to the Milk Production Traits of Slovak Spotted Cattle

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