Genetic Structure and Phylogenetic Relationships of the Polish Heavy Horse

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. Sequence alignment loci (in an ideal population, e ( u) should equal H H was performed by using a reference equine o), and ettl gives the total measure of expected mtDNA sequence (Xu and Arnason 1994). heterozygosity for blood protein loci. In addition, Gene frequencies for blood protein were calcu- populational inbreeding level was estimated by F H H lated by direct counting. Frequencies of alleles at Wright’s inbreeding coefficient is =1–( o/ e).

Table 1. Estimates of genetic variability for the Polish Heavy Horse and other selected domestic horse breeds. N = number of animals tested; Ho, He, Hu = observed, Hardy-Weinberg expected, and unbiased expected heterozygosity; Fis = inbreeding coefficient; Hettl = expected heterozygosity for 17 blood protein loci; Ae = effective number of alleles; Na = total number of identified variants.

Breed NHo He Hu Fis Hettl Ae Na Arabian 117 0.307 0.327 0.328 0.061 0.376 2.132 67 Belgian Draft 82 0.427 0.415 0.418 –0.028 0.451 2.386 66 Breton 32 0.387 0.352 0.357 –0.102 0.413 2.302 54 Clydesdale 41 0.334 0.322 0.326 –0.039 0.347 2.121 54 Friesian 314 0.307 0.306 0.307 –0.003 0.348 1.901 54 Hackney Horse 40 0.333 0.377 0.382 0.119 0.438 2.491 64 Haflinger 161 0.405 0.412 0.413 0.016 0.458 2.704 69 Hucul 112 0.343 0.305 0.307 –0.123 0.391 2.246 60 Irish Draft 108 0.404 0.393 0.42 –0.026 0.457 2.663 70 Lipizzaner 98 0.335 0.324 0.325 –0.034 0.366 2.396 69 Noriker 28 0.361 0.365 0.372 0.013 0.443 2.681 65 Percheron 81 0.404 0.400 0.402 –0.01 0.45 2.692 68 Quarter Horse 168 0.396 0.393 0.394 –0.007 0.44 2.653 87 Suffolk Punch 122 0.438 0.432 0.434 –0.013 0.465 2.459 65 Thoroughbred 265 0.294 0.288 0.289 –0.019 0.325 2.009 64 Trakehner 58 0.302 0.295 0.297 –0.024 0.363 2.041 59 Wielkopolska 295 0.352 0.326 0.326 –0.082 0.376 2.229 64 Polish Heavy Horse 204 0.391 0.386 0.387 –0.012 0.438 2.582 78 Mean for 122 populations 0.371 0.365 0.371 –0.015 0.413 2.39 65.11 SD for 122 populations 0.048 0.043 0.043 0.065 0.039 0.25 11.04

He, Hu, Ho were calculated from the 10 protein polymorphism loci only. Hettl was calculated from all 17 loci. All breeds (except the Polish Heavy Horse) were tested at the University of Kentucky. 356 E. Iwañczyk et al.

Values of genetic variation of the Polish Heavy typical for those of the heavy drought horse breeds Horse were compared to those of 122 domestic and show no clear evidence of loss of variation. horse populations that have been tested at the Uni- Figure 1 shows the genetic relationship among versity of Kentucky (Cothran E.G., unpubl. data). horse breeds from restricted maximum likelihood The same measures of variation were calculated (RML) analysis of the gene frequency data at for microsatellite data by using all 12 loci. Genetic blood group and blood protein loci. The draught relationships between the Polish Heavy Horse and horses form a distinct cluster that pairs with the other domestic horse breeds based upon gene fre- true pony breeds. Within this ‘cold-blooded’ quencies were investigated by using restricted group, the Polish Heavy Horse clusters most maximum likelihood (RML) analysis with closely with the Posavina breed from Croatia and PHYLIP software (Felsenstein 1989). Sequence the Breton, and this group is in the cluster with the alignment was performed by using a reference Belgian Draft horse breed. The Breton and Bel- gian were used in the development of the Polish equine mtDNA sequence (GeneBank X79547). breed. The Belgian has been important in the origins of a number of heavy draft breeds and it is Results and discussion likely that all the heavy breeds share a common ancestral origin with later diversification. The po- sition of the Polish Heavy Horse breed within the At the blood group and blood protein loci, genetic tree is what would be expected for a recently de- variation for the Polish Heavy Horse was slightly rived breed. greater than the mean values for domestic horse Estimates of genetic variability for breeds (Table 1). Populational diversity measured microsatellite (mSat) loci for the Polish Heavy by Ae and Na was high, which probably reflects the Horse and other domestic horse breeds are given mixed breed origins of this Polish breed. Patterns in Table 2. Variation at protein loci was relatively of variation within the Polish Heavy Horse are higher than that based upon mSat loci, although in

Figure 1. Consensus tree from 20 RML trees comparing the Polish Heavy Horse to 66 domestic breeds and the Przewalski horse (used as an outgroup) Genetics and phylogeny of Polish Heavy Horse 357

Table 2. Genetic variability at microsatellite loci for the Polish Heavy Horse and other domestic horse breeds. N = number of animals tested. Ho, He, Hu = observed, Hardy-Weinberg expected, and unbiased expected heterozygosity; Fis = inbreeding coefficient; Ae = effective number of alleles; Na = total number of identified variants, MNA = mean number of alleles per locus.

Breed NHo He Fis Hu Ae Na MNA Akhal Teke 84 0.674 0.711 0.052 0.715 3.839 91 7.583 American Saddlebred 101 0.706 0.702 –0.005 0.706 3.858 89 7.417 Arabian 22 0.645 0.646 0.002 0.661 3.339 64 5.333 Dales Pony 53 0.710 0.656 –0.082 0.663 3.335 68 5.667 Exmoor Pony 98 0.601 0.606 0.008 0.609 2.732 63 5.250 Fell Pony 53 0.781 0.723 –0.080 0.730 3.967 77 6.417 Friesian 199 0.452 0.441 –0.025 0.442 2.013 59 4.917 Haflinger 341 0.692 0.639 –0.082 0.640 3.264 75 6.250 Hanoverian 26 0.700 0.750 0.066 0.765 4.255 79 6.583 Holstein 21 0.769 0.679 –0.131 0.696 3.359 63 5.250 Hucul 127 0.667 0.712 0.063 0.715 4.042 91 7.583 Polish Primitive Horse 158 0.652 0.664 0.019 0.667 3.544 73 6.083 Lippizanner 150 0.686 0.698 0.018 0.701 3.419 89 7.417 Lithuanian Heavy Drought 24 0.759 0.718 –0.058 0.733 3.936 75 6.250 Sella Francais 34 0.719 0.718 –0.001 0.729 3.836 72 6.000 Shetland Pony 36 0.670 0.662 –0.013 0.671 3.176 72 6.000 Suffolk Punch 115 0.678 0.726 0.066 0.729 3.972 81 6.750 Tennessee Walking Horse 32 0.660 0.653 –0.012 0.663 3.307 70 5.833 Thoroughbred 114 0.673 0.692 0.027 0.695 3.589 74 6.167 Zemaitukai (heavy type) 30 0.758 0.715 –0.061 0.727 3.783 69 5.750 Zemaitukai (old type) 31 0.682 0.641 –0.064 0.651 3.041 60 5.000 Polish Heavy Horse 448 0.691 0.717 0.036 0.716 3.981 97 8.083 Mean for 55 populations 0.699 0.69 –0.013 0.706 3 682 73.8 6.147 SD for 55 populations 0.062 0.058 0.056 0.061 0.526 12.9 1.081

All breeds (except the Polish Heavy Horse) were tested at the University of Kentucky absolute values the mSat variation is greater. Indi- heavy horses and ponies and shows the closest vidual genetic variation, as measured by observed similarity to Friesians, which could be attributed heterozygosity (Ho), was slightly above the mean to the limited number of heavy horse breeds typed numbers for domestic horse breeds. For popula- at microsatellite loci. tion variation measures (He, Ae and Na) all were Three different haplotypes were observed out higher than means for domestic horse breeds. of three randomly selected samples, which is simi- Na was especially high but this is probably due to lar to other findings: 13 haplotypes in 16 maternal the large number of animals typed. Figure 2 shows lines of Lipizzan horses (Kavar et al. 1999) and the RML tree generated from microsatellite data. 27 haplotypes in 34 Arabian maternal lines Not all the breeds used in the tree generated from (Bowling et al. 2000). Two of the Polish Heavy the blood group typing and blood protein polymorphism data had microsatellite data available Horse mtDNA haplotypes were the same as those and thus could not be used in the RML analysis, observed in the Irish Draft breed. One haplotype and only those breeds that fit within the was shared among Polish Heavy, Posavina and cold-blooded horse group plus the Polish Primi- Irish Draft. Despite the haplotype similarity to the tive Horse are shown in Figure 2. The Polish Irish Draft, this breed does not show any close re- Heavy Horse is placed between the clusters of lationship to the Polish Heavy Horse for nuclear 358 E. Iwañczyk et al.

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