24th Int. Symp. “Animal Science Days”, Ptuj, Slovenia, Sept. 21st−23rd, 2016. Acta argiculturae Slovenica, Supplement 5, 45–49, Ljubljana 2016 COBISS: 1.08 Agris category code: L10 GENETIC DIVERSITY OF OLD KLADRUBER AND NONIUS HORSE POPULATIONS THROUGH MICROSATELLITE VARIATION ANALYSIS Nina MORAVČÍKOVÁ 1, Radovan KASARDA 2, Veronika KUKUČKOVÁ 3, Luboš VOSTRÝ 4, Ondrej KADLEČÍK 5 Genetic diversity of Old Kladruber and Nonius horse populations through microsatellite variation analysis 1 Slovak University of Agriculture in Nitra, Faculty of Agrobiology and Food Resources, Department of Animal Genetics and Breeding Biology, Tr. A. Hlinku 2, 94976 Nitra, Slovakia, e-mail: nina.moravcikova@uniag.sk 2 Same address as 1, e-mail: radovan.kasarda@uniag.sk 3 Same address as 1, e-mail: veron.sidlova@gmail.com 4 Department of Genetics and Breeding, Czech University of Life Sciences Prague, Kamycka 129, 165 21 Praha 6-Suchdol, the Czech Republic, e-mail: vostry@af.czu.cz 5 Same address as 1, e-mail: ondrej.kadlecik@uniag.sk ABSTRACT The aim of this study was to evaluate the genetic diversity across horse populations of Old Kladruber and Nonius breeds and to assess the degree of subdivision based on genetic relationships within them. In total, 270 Czech Old Kladruber and 18 Nonius horses from Slovak studs have been included in study. To determine the level of genetic vari- ability the dataset of 17 microsatellites has been used. The average values of observed heterozygosity (0.68 ± 0.03) and gene diversity (0.66 ± 0.02) indicated good level of variability across populations. The value of FIS (0.04 ± 0.02) showed relative balanced proportion of homozygotes and heterozygotes and signalized the existence of HWE across popula- tions. According to the value of Shannon’s information index (1.48 ± 0.07) the high degree of overall variability has been confirmed. Most of the variations in dataset explained the differences among individuals within each population. The FST index and Nei’s distances showed stronger connection between the Old Kladruby black variety and the Nonius, compared to the grey variety. The segregation of Nonius and Old Kladruber populations demonstrated also results of PCA analysis. Key words: horses, breeds, Old Kladruber, Nonius, genetic diversity, genetic variability, microsatellite markers 1 INTRODUCTION The management of genetic diversity within popula- tion is a key factor in any breed conservation programme for protecting the animal genetic resources (Gupta et al., 2015). Domestication of livestock species including horse populations and long history of migration, selection, and adaptation within them have created an enormous vari- ety of breeds (Groeneveld et al., 2010). Over the last sev- eral centuries, more than 400 distinct horse breeds have been established by genetic selection and have held a valuable place within other livestock species through ser- vice in war, agriculture, sport and companionship (Mc- Cue et al., 2012). Changes of conditions in production systems as well as significant competition among breeds create expectations of risks, which will negatively influ- ence their surviving and can result in the loss of variabil- ity through stochastic sampling, particularly when the number of reproducing individuals is restricted (Kasarda and Kadlečík, 2007; Winton et al., 2015). Based on the fact that the ancient old Spanish horses are extinct, the Old Kladruber breed is unique but also endangered by the loss of diversity, mainly due to the historical bottlenecks and intensive inbreeding (Petla- chová et al., 2012; Janova et al., 2012). The Old Kladruber horse originates from old Spanish and Italian bloodlines in the sixteenth and seventeenth century. It was bred as the native karst horse, and during succeeding genera- tions it was crossed with the old Neapolitans and horses of Spanish descent obtained from Spain, Germany, and Acta agriculturae Slovenica, Supplement 5 – 201646 N. MORAVČÍKOVÁ et al. The characterization of genetic diversity state with- in populations was realised by the calculation of fol- lowingparameters: the mean number of alleles (MNA), observed heterozygosity (Ho), gene diversity expressed as expected heterozygosity (He), effective allele number (Ane) and Shannon’s information index (I) using Genalex version 6.1 (Peakall and Smouse, 2006, 2012). The de- parture from the Hardy-Weinberg equilibrium (HWE) resulting from significance of differences between ob- served and expected genotype frequencies was tested by the Chi-square test. The amount of inbreeding-like effect across (FIT) and within (FIS) populations was evaluated according to Weir and Cockerham (1984). An analysis of molecular variance (AMOVA) estimating the genetic structure indices using information on the allelic content of haplotypes, as well as their frequencies stored entered as a matrix of Euclidean squared distances, was per- formed using 10,000 permutations with Arlequin v3.5 software (Excoffier et al., 2005). The genetic differentia- tion among populations reflected relationship within in- dividuals as well as populations were estimated based on the Nei’s distance and Wright’s FST index using Genalex version 6.1. Subsequently, the subdivision of each pop- ulation was tested based on the principal components analysis (PCA) adopted in R package Adegenet (Jombart and Ahmed, 2011) and visualised by R software v 3.2.2 (R Core Team, 2013). 3 RESULTS AND DISCUSSION Across selected populations and analysed mic- rosatellite markers the total number of alleles 132 was found. The numbers of alleles ranged from 5 (HMS1) to 12 (ASB17) and was in average 7.76 ± 0.45. The effec- tive allele numbers ranged from 6.85 (VHL20) to 1.99 (HTG6). The value of observed average heterozygosity (0.68 ± 0.03) indicated a good level of variability across individuals. The Ho was in range from 0.5 (HTG6) to 0.86 (VHL20). Based on gene diversity the same state of vari- ability was found. The He varied from 0.49 (HTG6) to 0.85 (VHL20). The sufficient proportion of heterozygous animals was indicated also by FIS index. Across all loci the average FIS value close to zero (0.04 ± 0.02) indicated rel- atively balanced proportion of heterozygotes vs. homozy- gotes and also signalise the HWE in populations. How- ever, using Chi-square test the departure from HWE due to the significance of differences between observed and expected genotype frequencies (P < 0.05) was identified at up to 12 loci (AHT4, AHT5, ASB17, ASB23, HMS2, HMS3, HMS6, HMS7, HTG6, HTG7, LEX3, VHL20). According to Shannon’s information index the lo- cus VHL20 (I = 1.96) was considered as the most in- Denmark (Lynghaug, 2009). The Old Kladruber horse has also played a part in the development of other horse breeds, for example Nonius and Lipizzan. The Kladruby horses were bred at Mezöhegyes stud in Hungary, and were one of the bases of the Nonius breed which takes name from its foundation sire a French stallion known as Nonius Senior. Born in Normandy in 1810, Nonius Senior was a  mixed breed, with Norfolk Roadster and Norman blood. Nonius Senior was bred to mares of sev- eral different breeds, and his descendants were interbred similarly with Spanish Neapolitan horses and later, for further refinement, with English Thoroughbreds. Today, the steady and stoutly built Nonius is used under saddle and in harness (Reeve and Bigs, 2011). The comprehensive information about genetic di- versity and population structure is highly important to evaluate the essential outlines for any appropriate con- servation and sustainable management of breeding programs to preserve maximal genetic variability and future adaptation potential of breeds (Khanshour et al., 2015; Winton et al., 2013). The molecular genetic studies of diversity regarding the horse populations are largely based upon microsatellite markers which offer advan- tages that are particularly appropriate for conservation projects (Bordonaro et al., 2012). Microsatellites are used as highly informative markers in population analysis be- cause they allow the detection of distorting events in the population (selection, migrations, random drift) as well as the degree of inbreeding (Dovč et al., 2006). The aim of this study was to determine the level of genetic diversity based on microsatellite variability with- in and across populations of Old Kladruber and Nonius horses. Besides that the degree of potential populations’ subdivision and relationship resulting from historical connectedness between the breeds have been evaluated. 2 MATERIAL AND METHODS In this study the genotyping data obtained from 238 horses of two breeds, Old Kladruber (270) originating from Czech Republic and Nonius (18) from Slovak studs were evaluated. For description of population subdivi- sion within the Old Kladruber horses two colour varie- ties have been considered as separate populations (grey variety, 175 animals; black variety, 95 animals). To de- termine the level of genetic variability within and across each selected horse population the dataset of 17 micros- atellite loci (AHT4, AHT5, ASB17, ASB2, ASB23, CA425, HMS1, HMS2, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, HTG10, LEX3, and VHL20), recommended pri- marily for paternity testing by ISAG (International Soci- ety for Animal Genetics) has been used. Acta agriculturae Slovenica, Supplement 5 – 2016 47 GENETIC DIVERSITY OF OLD KLADRUBER AND NONIUS HORSE POPULATIONS ... MICROSATELLITE VARIATION ANALYSIS formative. The average value of I index across all loci (1.48 ± 0.07) also reflected high degree of overall genetic variability within populations, because the Shannon’s in- formation index generally reflected the effectiveness of microsatellite markers to reveal the genetic variations. Figure 1 shows the distribution of four basic genetic pa- rameters describing the state of diversity across selected horse populations for each locus. Overall, all of the ap- plied genetic diversity parameters expressed good level of genetic variability within evaluated populations (Table 1) and indicated the state of biodiversity comparable with studies assessed Hungarian Noric population (Mihók et al. 2009), Czech Old Kladruber (Vostrý et al., 2011) and also other horse breeds with Austrio-Hungarian ori- gin (Achmann et al., 2004; Dovč et al., 2006). The AMOVA analysis has been applied to evaluate the hierarchical population structure and to explain the proportion of differences affected by the subdivision of populations. The results showed that the most of the vari- ation was explained by the differences conserved among individuals within each population (84 %). The subdivi- sion of populations according to their origin reflected up to 9 % of the genetic diversity in the analysed dataset and 7 % of total genetic variability was partitioned within in- dividuals in whole population. The obtained values of FST index and Nei’s genetic distances as frequently used indicators of relatedness significantly confirmed the connectedness between ana- lysed populations resulting from their historical origin. According to the generally accepted criteria the average value of FST at level 0.08 can be regarded as low and the populations as only slightly genetically differentiated. But the FST values at level 0.03 also indicated expected closest genetic similarity between both the Old Kladruber pop- ulations. Based on our results, the connection between the Old Kladruby black variety to the Nonius seems to be relatively stronger (0.05) compared to the grey vari- ety (0.07), which showed also subsequent evaluation of Nei’s genetic distances. At the intra-population level of Old Kladruber the DA value 0.17 was found. The highest differentiation was identified between the Old Kladruber grey variety and population formed by the Nonius horses (DA = 0.41). The clear segregation of Nonius and Old Kladruber populations was demonstrated also based on PCA analy- sis. Moreover, this analysis showed the separation of grey Figure 1: Distribution of effective allele numbers (A), observed heterozygosity (B), Wright’s FIS index (C) and Shannon’s information index (D) per locus (in order AHT4, AHT5, ASB17, ASB2, ASB23, CA425, HMS1, HMS2, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, HTG10, LEX3, and VHL20) across analysed population. Population Ane I Ho He F Nonius 4.05 ± 0.28 1.46 ± 0.08 0.82 ± 0.02 0.73 ± 0.02 −0.39 ± 0.05 Old Kladruber black 3.48 ± 0.29 1.40 ± 0.07 0.67 ± 0.04 0.68 ± 0.03 0.02 ± 0.02 Old Kladruber grey 3.31 ± 0.26 1.32 ± 0.08 0.65 ± 0.04 0.66 ± 0.03 0.01 ± 0.04 Table 1: The average values of genetic diversity parameters obtained across microsatellites Acta agriculturae Slovenica, Supplement 5 – 201648 N. MORAVČÍKOVÁ et al. and black Old Kladruber colour variety (Fig. 2). First two principal components were sufficient for the explanation of the genetic structure and division of individuals in to the groups. The PC1 component explained 18.52  % of the variation, while the second components explained 7.97 % of the variability. Preservation of endangered species is one of the most important goals for the present biological sciences, especially in the context of natural ecosystem stability. In the case of horse, conservation programs are usually initiated for breeds which present a unique genetic and phenotypic value (Mackowski et al., 2015). An under- standing of the genetic characteristics of a population is paramount in order to inform and implement conserva- tion strategies that maintain genetic diversity (Winton et al., 2013). The baseline molecular analysis provides a dependable tool which can be used together with the quantitative approach and traditional breeding strategies for an efficient design of preservation strategy. Moreo- ver, the genetic distances can be used for explanation of the population structure and genetic distinctiveness of breeds (Gupta et al., 2015). 4 CONCLUSION Our study showed that the level of biodiversity con- served within both analysed horse populations estimated using 17 microsatellite systems, is sufficient. The genetic differentiations between the Old Kladruber and Nonius population clearly reflected the formation of separate groups in accordance to their breeding history. The re- sults also showed that the connection between the Old Kladruby black variety to the Nonius seems to be rela- tively stronger compared to the grey variety that reflect- ed mainly the breeding background of Nonius horses. 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