MICROSATELLITE DIVERSITY OF CROSSBRED HORSES RAISED IN BOSNIA AND HERZEGOVINA

The focus of this study was microsatellite diversity of crossbred horses raised in Bosnia and Herzegovina. Genomic DNA was extracted from blood samples of 20 individuals (KBA group – 7 individuals, crosses between Bosnian and Herzegovinian mountain horse and Arabian horse; KBR group – 9 individuals, crosses between Bosnian and Herzegovinian mountain and Belgian horses, crosses between Bosnian and Herzegovinian mountain horses and Holstein, crosses between Bosnian and Herzegovinian mountain and Lipizzaner horses and KBN group – 4 individuals, crosses between Bosnian and Herzegovinian mountain horse with an unknown origin of the other parent). The samples were profiled using 17 microsatellite markers. This method consisted of multiplex PCR procedure and generated reasonable amplification across all the loci. All samples were genotyped successfully. Considering all the observed parameters, VHL20 locus showed the highest microsatellite diversity. Locus HMS7 was the least variable in KBR group, while HMS1 locus was the least diverse in KBN group. The highest microsatellite diversity in KBA group was found at AHT5 locus while HTG6 locus was the least diverse. Obtained results suggest that the investigated populations of crossbred horses from Bosnia and Herzegovina are not affected by substantial loss of genetic diversity, as indicated by the presence of reasonably high level of genetic variation. An increase in the inbreeding coefficient and sufficient heterogenity in KBN group indicate occurrence of consanguineous mating. The present research contributes to the knowledge of current status of genetic structure of the investigated crossbred horses.


Introduction
Microsatellites (Short Tandem Repeats -STR) are a class of genetic markers, currently the most commonly used for diversity studies in livestock (Fornal et al., 2013;Semik and Zabek, 2013).Due to their high level of polymorphism, dispersion throughout eucariotic nuclear genome and Mendelian co-dominant inheritance, microsatellites are relatively easy to score and are considered the markers of choice in equine parentage testing and individual identification (Zabek and Fornal, 2009;Moshkelani et al., 2011).Microsatellites are regions of repeated 2 to 7 nucleotide long units that occur primarily in non-coding regions of DNA (Moshkelani et al., 2011).Microsatellites have been employed to construct linkage maps, to examine population genetic structure and genetic variation, to explore molecular evolution, in studies of gene flow, in resolution of forensic cases and as parentage testing markers (Tozaki et al., 2003;Moshkelani et al., 2011).Microsatellites loci constitute an informative source concerning population history, structure and genetic diversity and microsatellite polymorphism still plays an important role in the assessment of genetic diversity of livestock (Semik and Zabek, 2013).
In this paper we report the results of the first analysis of microsatellite diversity of crossbred horses from Bosnia and Herzegovina (B&H) using 17 microsatellite markers currently recommended by International Society for Animal Genetics (ISAG) (ISAG, 2014).Number of different alleles, observed and expected heterozygosity, polymorphic information contents, inbreeding coefficient and deviation from Hardy-Weinberg equilibrium were estimated.

Materials and methods
The study was performed on 20 blood samples of horse crossbreeds (KBA group -7 individuals, crosses between Bosnian and Herzegovinian mountain horse and Arabian horse; KBR group -9 individuals, crosses between Bosnian and Herzegovinian mountain and Belgian horses, crosses between Bosnian and Herzegovinian mountain horses and Holstein, crosses between Bosnian and Herzegovinian mountain and Lipizzaner horses and KBN group -4 individuals, crosses between Bosnian and Herzegovinian mountain horse with an unknown origin of the other parent).All horses were raised in Bosnia and Herzegovina (Rukavina et al., 2015b).Blood samples were collected from v. jugularis using sterile venipuncture needles and EDTA vacuum containers.Genomic DNA was isolated using salting-out method that was originally developed for the isolation of DNA from human blood (Miller et al., 1988).Necessary modifications to the protocol were made in order to accommodate for different properties of horse blood as well as our laboratory conditions (3ml of blood; 10ml of Lysis buffer; 4ml of PBS; 4ml of Kern-lysis buffer; 150μl of 20% SDS; 100μl of protease and 0,5ml 6M NaCl).In total, 20 animals were genotyped for 17 microsatellite loci.This method consists of multiplex PCR procedure and shows satisfactory amplification of all analyzed fragments.Fragment separation and allele sizing were performed using ABI Prism 310 Genetic Analyzer.All the genotypes were successfully generated.Sizing of the amplified fragments was performed using GeneMapper ID v3.2 software.Number of different alleles (AN), polymorphic information content (PIC) (Botstein et al., 1980), observed heterozygosity (Ho), expected heterozygosity (HE) (Nei, 1987), inbreeding coefficient (F) (Weir, 1996) and deviation from Hardy-Weinberg equilibrium (HWE) (Guo and Thompson, 1992) was calculated using POWERMARKER 3.25 (Liu and Muse, 2005).

Conclusions
The results of the present study suggest that the investigated populations of crossbred horses raised in B&H are not affected by major loss of genetic diversity.The applied set of 17 microsatellite markers proved to be specific enough for use in study of genetic structure of crossbred horses.An increase in inbreeding coefficient and sufficient heterogenity between animals in KBN group indicate occurrence of consanguineous mating.The present research contributes to the knowledge of population structure and current status of genetic structure of the investigated populations.Also, the results offer basic information that may be helpful to horse breeders in designing and managing future breeding strategies.

Table 1 .
. In KBR group the mean Number of alleles (AN), expected heterozygosity (HE), observed heterozygosity (HO), polymorphic information content (PIC), inbreeding coefficient (F) and deviation from Hardy-Weinberg equilibrium (HWE) at 17 microsatellite loci in KBR group