Evaluation of the 17-PLEX STR kit for parentage testing of Polish Coldblood and Hucul horses.

in this study, 17 microsatellite markers were evaluated for parentage testing in a group of hucul and polish Coldblood horses, whose parents were excluded in single loci. despite the high polymorphism of the majority of 17 microsatellites an increase in homozygosity and deviations from h-W proportions were observed in some loci in the investigated populations. deviations were observed mainly in loci where single exclusions were found. dna typing problems of some microsatellite sequences, sex linkage of markers and the occurrence of null alleles are the basis for erroneous parentage exclusion. the relationship between these issues and population statistics and their impact on efficiency of parentage testing is discussed. key words: horses, microsatellites, parentage testing, single-locus exclusion Short Tandem Repeat (STR) loci, i.e. microsatellites, are a class of nuclear DNA markers consisting of tandemly repeated sequence motifs of two to seven base pairs in length. Alleles of STR loci vary by the number of times a given sequence motif is repeated. STR alleles are detected using Polymerase Chain Reaction (PCR) and by separating the amplification products using electrophoresis. Due to their high level of polymorphism (informativeness) and Mendelian inheritance, microsatellites have become the markers of choice for parentage testing and individual identification. In Poland, the polymorphism of these markers has been proved to be useful in purebred Arabian (Gralak et al., 1998) and Anglo-Arabian (Ząbek et al., 2006), Thoroughbred (Gralak et al., 1998; Niemczewski and Żórkowski, 2000; Ząbek et al., 2003) and Silesian (Ząbek et al., 2003), Polish Primitive (Gralak et al., 2001), Polish Heavy (Iwańczyk et al., 2006) and Biłgoraj horses (Ząbek et al., 2005). Combining a set of more than 7 highly informative microsatelite markers in one multiplex reaction can exclude wrongly determined parentage with probability greater than 99% (Ząbek, 2006). Some problems occur during application of STR systems in routine parentThis work was conducted as part of the NRIAP statutory activity, project no. 3122.1. T. Ząbek and A. Fornal 364 age testing. Proper designation of STR alleles is disturbed by nonspecific addition of adenine to the 3’ end of the PCR products (plus A product) and by mutations in the primer-binding site, which prevent the amplification of one of the alleles in a microsatellite locus (null allele) (Eggleston-Stott et al., 1997). Cases when only one locus excludes the alleged parentage are the common signs of related STR typing problems. Therefore it was assumed that exclusion is validated if inconsistency in genetic basis between offspring and alleged parent will be discovered in at least two microsatellite loci. In this work we discuss the use of 17 microsatellite markers for parentage testing in a group of Hucul (hc) and Polish Coldblood (pcb) horses, which represent cases where single-locus exclusions of one of their parents were detected. Population of individuals sampled from such difficult parentage cases was considered to be appropriate for finding the reasons of single exclusions at particular loci. The STR panel encompasses the following equine loci: VHL20, HTG4, AHT4, HMS7, HTG6, AHT5, HMS6, ASB23, ASB2, HTG10, HTG7, HMS3, HMS2, ASB17, LEX3, HMS1 and CA425. These include nine loci recommended by the Equine Genetics and Thoroughbred Parentage Testing Standardization Committee of the International Society for Animal Genetics (ISAG) and eight additional loci commonly used for horse parentage testing and identification worldwide (Table 1). Evaluation was made of the quality of DNA profiles of the investigated microsatellite loci, deviations from Hardy-Weinberg proportions in relation to the differences between observed and expected heterozygosity for particular marker loci, and the frequency of null alleles. Table 1. The list of microsatellite markers and chromosomal location Locus Chromosome Literature AHT4* 24 Binns et al. (1995) AHT5 8 Binns et al. (1995) ASB17 2 Breen et al. (1997) ASB2 15 Breen et al. (1997) ASB23 3 Irvin et al. (1998) HMS1 15 Guerin et al. (1994) HMS2 10 Guerin et al. (1994) HMS3 9 Guerin et al. (1994) HMS6 4 Guerin et al. (1994) HMS7 1 Guerin et al. (1994) HTG10 21 Marklund et al. (1994) HTG4 9 Ellegren et al. (1992) HTG6 15 Ellegren et al. (1992) HTG7 4 Marklund et al. (1994) LEX3 X Coogle et al. (1996) UCDEQ425 28 Eggleston-Stott et al. (1997a) VHL20 30 Van Haeringen et al. (1994) ISAG recommended markers for parentage testing are in bold. Microsatellite markers for parentage testing of horses 365 Material and methods The studied populations of horses comprised 195 males and 29 females of hc horses and 70 males and 6 females of pcb horses. All individuals represent cases where inconsistency between parental and offspring genotype was only detected in a single locus. DNA was prepared from 300 μl of blood using Wizard kit (Promega) according to the manufacturer’s protocol. The final DNA concentration was in the range of 20 to 60 ηg/μl. 2 μl of DNA were amplified in one multiplex reaction using a commercial kit from Finnzymes Diagnostic (Finnland), which includes the PhusionTM hot start DNA polymerase with proofreading activity and fluorescently labelled primers specific for 17 microsatellite loci (Table 1). PCR products were separated in a capillary electrophoresis system on a 3130xl genetic analyser (Applied Biosystems). Each sample was run with size standard LIZ 500, applied for automated DNA fragment analysis with the use of five fluorescent dyes. Fragment analysis and verification of DNA profiles for 17 STR loci were performed in the Genemapper 4.0 software. The calculation of allele frequency, observed (Ho) and expected heterozygosity (He) (Nei, 1978), polymorphism information content (PIC) (Botstein et al., 1987) and probability of exclusion (PE) of wrongly assigned parentage when one and both parental genotypes are known (Jamieson and Taylor, 1997) were performed using the CERVUS 2.0 program. This package was also used to perform the Chi square test for the presence of Hardy-Weinberg equilibrium (H-W). The frequency of null alleles was estimated for each marker locus, using an iterative algorithm based on the difference between the observed and expected frequency of homozygotes (Summers and Amos, 1997) (CERVUS 2.0 package).