J Appl Genet 47(4), 2006, pp. 353–359 Original article Genetic structure and phylogenetic relationships of the Polish Heavy Horse Ewa Iwañczyk1, Rytis Juras2, Grzegorz Cholewiñski1, E. Gus Cothran2 1Horse Genetic Markers Laboratory, August Cieszkowski Agricultural University of Poznañ, Poland 2Texas A&M University, College Station, Texas, USA Abstract. In this study a wide range of genetic markers (12 microsatellites, 7 blood-group loci, 10 blood-protein loci) and mitochondrial DNA (mtDNA) were used to assess genetic diversity in Polish Heavy horses. Three ran- dom samples were sequenced for 421 bp of the mitochondrial D-loop region, but no clear phylogenetic patterns were seen in mtDNA variation. Both heterozygosity and diversity levels are fairly high in Polish Heavy horses. In phylogenetic analysis the draught horses form a distinct cluster that pairs with the true pony breeds. Within this ‘cold-blooded’ group, the Polish Heavy Horse clusters most closely with the Posavina breed from Croatia and the Breton breed from France. From the standpoint of genetic conservation, the Polish Heavy Horse does not appear to be in jeopardy. Key words: blood groups, genetic variation, phylogenetic relationship, mitochondrial DNA, Equus caballus, Polish Heavy Horse. Introduction Here we report the results of the first genetic analysis of the Polish Heavy Horse. Heavy horses As with the breeds of other domestic species, started to be imported to Poland in the second half some horse breeds are threatened by extinction be- of the 19th century. These were mainly stallions of cause they do not appear to meet current needs. the Percheron, Breton and Ardens breeds. Until af- Genetic characterization of populations can be ter World War II, no organized breeding of heavy - a useful first step in breed conservation and may horses existed in Poland in any practical sense. Af ter World War II, there was a great need for horses have implications for future breeding strategies to be used in field work on small farms in Poland. and management plans. Genetic markers designed The draught power of horses was also used for for parentage verification have been extensively transportation at that time. In 1946–1947 over - used to assess levels of genetic variation of differ 100 000 horses were delivered to Poland by the ent horse populations, to compare populations and UNRRA (United Nations Relief and Rehabilita- to determine relationships with other populations tion Administration). Another 50 000 horses were Z (Cothran and Van Dyk 1998; Ca on et al. 2000; imported from Belgium, Denmark, Finland, Bjrrnstad et al. 2000; Juras et al. 2003; Tozaki the Netherlands, Norway and Sweden. As a result et al. 2003; Aberle et al. 2004). As well as genomic of breeding work, a few local types (Lowicki, DNA, mitochondrial DNA is useful for studying Sztumski, Lidzbarski and Sokolski horse) were the evolution of closely related species. Many developed. studies have focused on the mitochondrial D-loop The current population of the Polish Heavy region, the most variable part of mtDNA (Ishida Horse derives mainly from crosses of local mares et al. 1994) due to a higher substitution rate than in with stallions of ‘cold-blooded’ breeds, such as the rest of mtDNA genome (Cann et al. 1984). French Arden, Swedish Arden, Belgian, Breton, Received: September 15, 2006. Accepted: October 16, 2006. Correspondence: E. Iwañczyk, Horse Genetic Markers Laboratory, August Cieszkowski Agricultural University of Poznañ, Wo³yñska 33, 60–637 Poznañ, Poland; e-mail: [email protected] 354 E. Iwañczyk et al. Rhinelander, Dole, North-Swedish and Soviet in accordance with the internationally standard- Heavy Horse. Although the need for the horses in ized usage for horses (Bowling and Clark 1985; agriculture has decreased in recent times, they still Bowling and Ryder 1987), except for variants at have a role on small farms. Currently, some loci, which have not yet received interna- ‘cold-blooded’ horses make up about 50–60% of tional recognition. the total horse population (400 000 animals) in Po- The DNA typing panel consisted of 12 AHT4 AHT5 land. In addition to their use for draught power, microsatellites: and (Binns et al. ASB2 HMS2 HMS3 heavy horses have an important role as slaughter 1995), (Breen et al. 1997), , , HMS6 HMS7 HTG4 animals for export to West Europe and are an im- and (Guérin et al. 1994), and HTG6 HTG7 HTG10 portant source of income for small farmers. (Ellegren et al. 1992), and VHL20 The aim of this study was to conduct a compar- (Marklund et al. 1994), and ative analysis of genetic diversity of the Polish (Van Haeringen et al. 1994). In the USA testing, Heavy Horse by using a wide range of genetic amplification of microsatellites in multiplex poly- markers and data from other domestic horse popu- merase chain reactions (PCRs) was performed in m lations. 25 L of total volume, containing 50 ng of genomic DNA, 0.07 to 0.8 pmol of each primer, 1´ PCR buffer (Perkin Elmer, MA, USA), 2.5 mM Materials and methods MgCl2, 0.2 mM dNTPs and l U AmpliTaq (PE Ap- plied Biosystems, MA, USA). For microsatellite Blood samples were collected by jugular amplification, a hot start procedure was used, in venipuncture in acid-citrate-dextrose (ACD) from which DNA template and primers were combined 204 horses for blood group and biochemical and heated at 95°C for 10 min. The temperature polymorphisms and from 448 horses for was then lowered and held at 85°C for 10 min for microsatellite polymorphisms. The samples were addition of the remaining reagents. This was fol- separated into red blood cells (rbc), rbc lysate, and lowed by 32 cycles of 1 min at 95°C, annealing at serum. Genetic analyses were done both in Poland 58°C for 30 s and 72°C for 45 s, and a final exten- and the USA and procedures for each lab (where sion at 72°C for 30 min. Reaction products were different) are given here. DNA was extracted from analysed by using an ABI Prism 377 DNA se- both whole blood and buffy coat with the quencer (Applied Biosystems, Foster City, CA, Puregene DNA Extraction Kit (Gentra Systems, USA). Fragment sizes were determined with the USA) following the manufacturer’s instructions computer software STRand (Hughes 2000). (USA, Lexington lab) and Chelex 100 (Sigma, Testing in Poland was similar except that it used USA) following the Walsh procedure (Walsh et al. 10 mL of total volume with 20 ng genomic DNA, 1991) (Poland, Poznañ lab). 1 × PCR buffer (Eurx, Gdañsk, Poland) and 1 U Standard immunological procedures involving polymerase (Eurx, Gdañsk, Poland). The PCR haemagglutination and complement-mediated standard procedure was a predenaturation step of haemolysis (Stormont and Suzuki 1964; Stormont 4 min at 94°C, followed by 30 cycles of 94°C for et al. 1964) were used to detect variation of red cell 30 s (denaturation), 60°C (annealing) for 30 s and alloantigens at 7 blood group loci. Starch and 72°C (extension) for 1 min, and a final extension polyacrylamide gel electrophoresis and isoelectric step of 10 min at 72°C. Reaction products were focusing were used to detect variation at 10 serum separated by using the ALFexpress II sequencer and rbc lysate protein loci (Braend 1973; (Pharmacia Biotech, Uppsala, Sweden) and frag- Sandberg 1974; Juneja at al. 1978; Braend and ment sizes were determined by using Fragment Johansen 1983; Pollitt and Bell 1980; Henney Analyzer 1.03 (Pharmacia Biotech, Uppsala, Swe- et al. 1994). den). All 448 samples were typed at the Poznañ The horse blood group loci examined were the lab, while 55 of them were typed in the USA. A, C, D, K, P, Q and U loci. The blood protein loci For mtDNA sequencing, primers from the pub- were a-1-b glycoprotein (A1b), albumin (AL), se- lished horse mtDNA sequence (Xu and Arnason rum esterase (ES), vitamin-D-binding protein 1994) were designed: forward 5’-CGCACA (GC), glucosephosphate isomerase (GPI), TTACCCTGGTCTTG-3’, reverse 5’- GAACCAGA alpha-haemoglobin (HBA), 6-phosphogluconate TGCCAGGTATAG-3’. PCR was carried out in dehydrogenase (6-PGD), phosphoglucomutase 25 mL of total volume, containing 0.2 mM (PGM), protease inhibitor (PI) and transferrin dNTP’s, 0.5 mM of each primer, 2.5 mM MgCl2, (TF). Nomenclature for variants at all 17 loci was 1 × PCR buffer,1UofTaqpolymerase (PE Ap- Genetics and phylogeny of Polish Heavy Horse 355 plied Biosystems, MA, USA),1UofAmpliTaq blood group loci were calculated by the allocation Gold (PE Applied Biosystems, USA), and 50 ng of method of Andersson (1985). Genetic variation template DNA. The reaction mixture was heated was measured as observed heterozygosity (Ho), to 95°C for 5 min, followed by 30 cycles, each Hardy-Weinberg expected heterozygosity (He), consisting of denaturation for 40 s at 94°C, anneal- unbiased expected heterozygosity (Hu) according ing for 45 s at 55°C, extension for 45 s at 72°C, and to Nei (1978), Hardy-Weinberg heterozygosity then a final extension for 10 min at 72°C. Se- from all 17 blood protein loci (Hettl), effective quencing was carried out by using a BigDyeTM number of alleles (Ae), and the total number of Terminator Cycle Sequencing Kit (PE Applied variants found in each population (Na). For the Biosystems, MA, USA) in an ABI Prism 377 blood protein loci, Ho was calculated for blood DNA Sequencer. Fragments of 421 bp each were protein loci only because of the presence of reces- sequenced but their length was truncated to 353 bp sive alleles and/or ambiguous genotypes at blood for phylogenetic comparison with the other horse group loci. Therefore, for direct comparison, breed sequences. All sequences were confirmed He and Hu were calculated only for blood protein by re-sequencing the same sample with a second H H independent PCR reaction.