The objective of the study was to determine the genetic distances and admixture in sire lines of Old Kladruber horse (populations in the context of a currently conducted conservation program) base on microsatellite analysis. Basic molecular parameters were estimated. The observed heterozygosity ranged from 0.48 to 0.85. The expected heterozygosity estimated for the sire lines was higher than observed heterozygosity. The minor genetic differences between sire lines of Old Kladruber horse were observed. Differences between the sire lines were identified using discriminant analysis. The first discriminant function separates the individuals of black and grey variants. For the discriminant function, however, the differentiation between sire lines was not so significant. These results showed that the breeding strategy revision is suggested in Old Kladruber horse

Alexandra Novotnà

Barbara Hofmanovà

Hana Vostrà-Vydrovà

Ivan Majzlik

Lubos Vostry

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287Agriculturae Conspectus Scientifi cus . Vol. 82 (2017) No. 3 (287-291)ORIGINAL SCIENTIFIC PAPERSummaryTh e objective of the study was to determine the genetic distances and admixture in sire lines of Old Kladruber horse (populations in the context of a currently conducted conservation program) base on microsatellite analysis. Basic molecular parameters were estimated. Th e observed heterozygosity ranged from 0.48 to 0.85. Th e expected heterozygosity estimated for the sire lines was higher than observed heterozygosity. Th e minor genetic diff erences between sire lines of Old Kladruber horse were observed. Diff erences between the sire lines were identifi ed using discriminant analysis. Th e fi rst discriminant function separates the individuals of black and grey variants. For the discriminant function, however, the diff erentiation between sire lines was not so signifi cant. Th ese results showed that the breeding strategy revision is suggested in Old Kladruber horse.Key wordspopulation structure; genetic diversity, endangered breedGenetic Distances and Admixture between Sire Lines of the Old Kladruber Horse Hana VOSTRÀ-VYDROVÀ 1Barbara HOFMANOVÀ 2Ivan MAJZLIK 2Alexandra NOVOTNÀ 1Lubos VOSTRY 1,2 ( )1 Institute of Animal Science, Přátelství 815, 104 01, Praha - Uhříněves, Czech Republic e-mail: vostry@af.czu.cz2 Czech University of Life Sciences Prague, Kamýcká 129, 16500, Praha 6 - Suchdol, Czech RepublicReceived: May 16, 2017 | Accepted: July 31, 2017ACKNOWLEDGEMENTS This work was financially supported through a grant from the Ministry of Agriculture of the Czech Republic (Project no. QJ1510141 and QJ1330189) and “S grant of MSMT CR”.Agric. conspec. sci. Vol. 82 (2017) No. 3288 Hana VOSTRÀ-VYDROVÀ, Barbara HOFMANOVÀ, Ivan MAJZLIK, Alexandra NOVOTNÀ, Lubos VOSTRYIntroductionOld Kladruber is the only horse breed that can be considered the original Czech breed that was bred especially for ceremonial purposes and aristocracy. Th e baroque character of the breed has been conserved until now. Th is breed is an important gene resource with unique characteristics and high cultural and his-torical value and has been continually kept in the territory of the Czech Republic for more than four hundred years. Th e origin of this breed dates back to 1579 on the Kladruby nad Labem stud farm. Th e main objective was to produce horses for the Imperial Court of the Hapsburgs (Bílek 1957). Th e Old Spanish horses, which were mainly imported from Italian stud farms, were used for this purpose. A decrease in breeding pop-ularity of the Old Spanish breed at the turn of the 18th and 19th centuries led to successive changes in the breeding program on the Kladruby nad Labem stud farm. Th e closed herd turnover began to be used, and it has been considered as a separate breed since then. Old Kladruber and Lipizzaner horses were princi-pally bred for the Imperial Court in Vienna on the same basis, i.e., Old Italian and Old Spain blood as the “baroque” horses with diff erent uses: Old Kladruber horses as carriage horses and Lipizzaner horses as riding horses. As a carriage horse, the Old Kladruber diff ers from the Lipizzaner in size, body conforma-tion and performance capacities. Affi  liation to the paternal line is one of the main criteria of a conservation program for the creation of the next generation parent couples. In a previous paper (Vostrá-Vydrová et al., 2016) we analyzed the genealogical information in order to ascertain the low level of genetic variability within the Old Kladruber horse. All results showed that the small genetic variability exist as the result of a small and closed population. Th e objective of this study was to determine the genetic dif-ferences between sire lines of the Old Kladruber horse based on microsatellite analysis.Material and methodsAnimal data collectionIn this study, 265 individuals from nine sire lines of Old Kladruber horse breed within a 38-year period (1978–2015) were used. Th e Old Kladruber horse lines, including the year of birth of the line founder, are described in Table 1. In the anal-ysis 17 animals of Generale sire line, 42 animals of Generale-Generalissimus sire line, 30 animals of Favory sire line, 22 animals of Favory-Generalissimus, 82 animals of Sacramoso sire line,18 animals of Rudolfo sire line, 30 animals of Solo sire line, 12 animals of Siglavi Pakra sire line and 30 animals of Romke sire line were included. Th e total set of 13 microsatellite markers (AHT4, AHT5, ASB2, HMS1, HMS2, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, HTG10 and VHL20) recommend-ed for parentage testing by the International Society for Animal Genetics (ISAG) and Equine Genetics Standing Committee was used for the analysis.Genetic diversityGenetic variability within populations was characterized as allele frequency, mean number of alleles, observed heterozygosity (HO), genec diversity, which is oft en called expected heterozygo-sity ( Weir, 1996),  testing of the Hardy-Weinberg equilibrium, the values of inbreeding, expressed as Wright’s fi xation indices – within a population (FIS) and between individuals within sub-populations (FST) were done using the PEGAS (Pa radis, 2010) package. Th ese indices provide more reliable results specifi cally for microsatellite data. Th e polymorphism information content (PIC; Bot stein et al., 1980) was calculated using PICcalc (Nagy, 2012).Population structure and genetic relationshipTo determine genetic structure and to infer genetic admix-tures, a discriminant analysis of principal components (DAPC) implemented in the Adegenet R package (Jo mbart and Ahmed, 2011) was used. Th e DAPC approach proposes an optimum dis-tribution of individuals into predefi ned groups in relation to the discriminant function of principal components. An optimum number of clusters was defi ned by the K-averaging algorithm that makes use of the Bayesian information criterion. In addi-tion, the DAPC was used to assign individuals and to obtain the membership probability which presents the overall genetic background of an individual. A trade-off  between the power of discriminant analysis and overfi tting of the given analysis was assessed by the α-score (Jo mbart and Ahmed, 2011). Results and discussionGenetic diversityEach of the analysed loci appeared as polymorphic, and their alleles were present in or shared by all studied sire lines. Th e total number of alleles found on 13 microsatellite markers across all sire lines was 92. Th e average number of alleles per locus was 7.08, ranging from 5 (HMS1) to 9 alleles (VHL20 and HTG10). Th e acquired PIC values were estimated from 0.289 (HTG7) to 0.78  Name  Colour Born Breed Origin Generale Generale - Generalissimus Favory Favory - Generalissimus Sacramoso Solo Siglavi Pakra Romke Rudolfo Gray Gray Gray Gray Gray/Black Black Black Black Gray 1787 1938 1779 1965 1800 1927 1946 1966 1968 Old Kladruber Old Kladruber Old Kladruber Old Kladruber Italo-Spanish Old Kladruber Lipizzaner Friesian Lusitanian Czech Republic Slovakia Slovakia  Czech Republic Italy Czech Republic Slovenia Netherlands Portugal Table 1. Name, colour variation, year of birth, breed and country of origin of the Old Kladruber horse line founderAgric. conspec. sci. Vol. 82 (2017) No. 3289Genetic Distances and Admixture between Sire Lines of the Old Kladruber Horse(VHL20). Th ese values indicate a relatively good polymorphism level according to Bot stein et al. (1980).  PIC is calculated with the total number of alleles and allele frequencies in a population. If PIC value is higher than value 0.75 the locus becomes much more informative. Th e observed heterozygosity between loci was from 0.47 (HTG7) to 0.85 (VHL20). Th e lowest values of genetic diversity were revealed in the HTG6 locus (0.47). Similarly, as for observed heterozygosity, the highest value of gene diversity was found in the VHL20 locus (0.85). Th e higher value of ex-pected heterozygosity then observed heterozygosity estimated in this study can be explained by the fact that there is mixing between sire lines due to alternative mating plan, which is used for the control of inbreeding using rotating mating between the lines. Overall information about diff erences and total statistics are shown in Table 2. Statistically signifi cant deviations from the Hardy-Weinberg equilibrium were found in more than two thirds of the microsatellite loci.All studied microsatellite markers showed high variability, and only in the markers ASB2, HMS2, HMS1 were not deviations from the Hardy-Weinberg equilibrium discovered. Generally, the genetic diversity of microsatellite loci can be aff ected by many factors, including genetic drift , impact of selective breed-ing, eff ect of individual stallions and random eff ects (Pe tersen et al., 2013). Th e determined values of genetic diversity were lower than, e.g., those in Polish cold-blood horses (Iw ańczyk et al., 2006) or Polish Konik (Sz waczkowski et al., 2016). Th e observed lower proportion of gene diversity distributed within autochthonous breeds can be explained mainly by the fact that the local breeds generally show higher degrees of diversity than breeds with more limited numbers of stallions and mares in the breeding scheme. Observed heterozygosity and the coeffi  cient of inbreeding measured by Wright’s FIS index, which are used as general indicators of the genetic diversity level, indicate a suf-fi cient level of heterozygosity in the studied population. Th ese results are not in agreement with the values of inbreeding co-effi  cient estimated by pedigree analysis. Th e average values of the inbreeding coeffi  cient estimated by pedigree analysis were 13% (with a maximum value of 29%) for the reference popula-tion (individuals that can currently take part in reproduction) (Vos trá-Vydrová et al., 2016). Th e molecular inbreeding meas-ured with a handful of molecular markers is not necessarily a good predictor of the genealogical or genomic inbreeding, that there are problems in estimation genomic heterozygosity using only a few molecular markers (Toro et al., 2009).Genetic structure and level of admixtureTo infer the population genetic structure and to assess the level of admixture, discriminant analysis of principal components was applied to genotyping data. Th e distribution of individuals according to the BIC analysis showed that inferred clusters do not correspond to actual groups (Figure 1). For this reason, the clusters in DAPC were inferred in line with the prior assump-tion of population distribution (K=10). Th e agreement between prior and posterior assignment was 78.10%. Based on the α-score (Jombart and Collins, 2015), which indicates the number of Locus NA PIC HO Gene diversity FST FIS HWE VHL20 9 0.784 0.8484 0.8517 0.0748 -0.0766 ** HTG4 7 0.707 0.7615 0.7732 0.0889 -0.0809 * AHT4 8 0.654 0.7558 0.8173 0.0831 -0.0086 ** HMS7 6 0.462 0.5850 0.6720 0.0927 0.0405 *** HTG6 6 0.153 0.4787 0.4719 0.0687 -0.0892 *** AHT5 7 0.484 0.7450 0.7505 0.0849 -0.0848 *** HMS6 6 0.487 0.7127 0.6842 0.0497 -0.0962 *** ASB2 8 0.720 0.8101 0.8039 0.0386 -0.0482  HTG10 9 0.611 0.6132 0.6611 0.0475 0.0262 * HTG7 6 0.289 0.4676 0.5525 0.1178 0.0405 *** HMS3 8 0.580 0.6058 0.6734 0.0440 0.0590 *** HMS2 7 0.662 0.7464 0.7387 0.0406 -0.0530  HMS1 5 0.330 0.5025 0.5443 0.0436 0.0347  where: * P<0.05, **P<0.01,***P<0.001 Table 2. Characteristics of 13 microsatellite loci analysed in nine sire lines (n=265). Number of successfully genotyped individuals (N), number of alleles (NA), polymorphism information content (PIC), observed heterozygosity (HO), gene diversity, Wright’s FST and HWE statisticsFigure 1. Genetic clusters determined using discriminant analysis of principal components where 1 – Generalle, 2 - Generale-Generalissimus, 3 - Favory, 4 - Favory-Generalissimus, 5 – Sacramoso, 6 – Solo, 7 - Siglavi Pakra, 8 – Romke, 9 - RudolfoAgric. conspec. sci. Vol. 82 (2017) No. 3290 Hana VOSTRÀ-VYDROVÀ, Barbara HOFMANOVÀ, Ivan MAJZLIK, Alexandra NOVOTNÀ, Lubos VOSTRYprincipal components (PCs) adjusted for the successful repeat-ed assignment of individuals, 26 PCA axes were left  in DAPC. Th ese axes correspond to more than 93.62% of variability. Th e two discriminant functions obtained correspond to 47.92% of variance. Th e fi rst discriminant function separates the individ-uals of black and grey variants (Figure 2). For  the discriminant function, however, the diff erentiation was not signifi cant (Figure 3). As expected, a certain level of admixture was revealed in all studied sire lines (Figure 4). Th e occurrence of individuals with high level of probability of admixture of other sire lines was de-termined in all sire lines. Th e results indicate that the DAPC is not sensitive enough to determine genetic diff erences between sire lines of the Old Kladruber horse.Th e results of DACP show that the structure of the studied sire lines of Old Kladruber horse was not suffi  ciently diff erenti-ated and that there was a high level of admixture between the sire lines. An admixture between the grey and black variants was proven, but to a limited extent. Th is admixture can be caused by the fact that individuals of the Sacramoso sire line are pre-sent in both colour varieties. Th e chosen methods were not able to correctly separate sire lines. Th ese results again refl ect the mating strategy in analysed breed. In such situation, this study might provide the fi rst insight into further conservation strate-gies that should be considered. ConclusionIn conclusion, this study presents a valuable insight into the genetic structure and diversity of sire lines of Old Kladruber horse. Our data suggest a low level of diff erentiation as well as a high gene fl ow between them. Hence, a revision of the breeding strategy is suggested. Th e modifi ed genetic conservation pro-gram should include both classical breeding methods based on pedigree information and molecular data.References Bílek F. (1957): Th e Old Kladruber Horse (CzechLanguage). In:Speciálnízootechnika II. Chovkoní, 74–104. Státnízemědělské Nakl, Praha, p.1031. Iwańczyk E., Juras R., Cholewiński G., Cothran E. G. (2006): Genetic structure and phylogenetic relationships of the Polish Heavy Horse. Journal of Applied Genetics, 47: 353–359.Figure 2. Genetic clusters determined using discriminant analysis based on the first discriminant function where 1 – Generalle, 2 - Generale-Generalissimus, 3 - Favory, 4 - Favory-Generalissimus, 5 – Sacramoso, 6 – Solo, 7 - Siglavi Pakra, 8 – Romke, 9 - RudolfoFigure 3. The BIC statistic results referring to differentiation between inferred and original clusters, where 10 – Generalle, 11 - Generale-Generalissimus, 12 - Favory, 13 - Favory-Generalissimus, 14 – Sacramoso, 15 – Solo, 16 - Siglavi Pakra, 17 – Romke, 18 - RudolfoFigure 4. Membership probability resulting from DAC analysis (K=10)Agric. conspec. sci. Vol. 82 (2017) No. 3291Genetic Distances and Admixture between Sire Lines of the Old Kladruber HorseJombart T., Ahmed I. (2011): adegenet 1.3-1: new tools for the anal-ysis of genome-wide SNP data. Bioinformatics, 27, 3070–3071.Jombart T., Collins C., 2015. A tutorial for discriminant analysis of principal components (DAPC) using adegenet 2.0.0. 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Genetic Distances and Admixture between Sire Lines of the Old Kladruber Horse

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