ISSN 1068�3674, Russian Agricultural Sciences, 2010, Vol. 36, No. 6, pp. 460–462. © Allerton Press, Inc., 2010.Original Russian Text © V.V. Kalashnikov, L.A. Khrabrova, A.M. Zaitsev, L.V. Kalinkova, M.A. Zaitseva, 2010, published in Doklady Rossiiskoi Akademii Sel’skokhozyaistven�nykh Nauk, 2010, No. 6, pp. 48–50.

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1 The occurrence of a large group of DNA markersopened new prospects for studying the characteristicsof the gene pool, origin, and microevolution of horsebreeds. Microsatellite markers are being used success�fully for marking genotypes and checking provenance[1–3]. Thanks to high variability, codominant inherit�ance, constancy in ontogeny, and known localizationin the genome, DNA microsatellites are ideally suitedfor studying the genetic characteristics and prove�nance of breeds [4, 5].

We made a comparative analysis of the allele pool ofimproved and local horse breeds, many of which arebred only in our country. The investigation includedtwo purebred breeds: Arabian and Thoroughbred, aswell as the most ancient improved breed of CentralAsia, the Akhal�Teke [6]. These three breeds had anenormous influence on the development of worldhorse breeding; therefore the task of our investigationsincluded a study of the characteristics and degree ofrelationship of these breeds at the molecular geneticlevel. During breeding of all purebred breeds, mixingwith horses of foreign blood was not allowed, whichwas fostered by the closed system of keeping studbooks, and presently a mandatory genetic verificationof origin. The Akhal�Teke breed, known already formore than 3000 years, undoubtedly influenced thecreation of the Arabian (VI–VII century AD), andhorses of eastern provenance participated in the for�mation of the gene pool of the Thoroughbred horse inthe first half of the XVIII century The characteristicsof polymorphism of microsatellite DNA of localbreeds, being a valuable genetic resource, were inves�tigated for the first time in the present investigation.

1 This work was supported by the Russian Foundation for BasicResearch (09�04�13725 ofi�ts.

METHOD

We used DNA samples extracted from the bloodand hair bulb of 12 horse breeds: Akhal�Teke (n =109), Arabian (n = 319), Thoroughbred (n = 443),Trakehner (n = 27), Standardbred (n = 77), Altai (n =29), Vyatka (n = 10), Buryat (n = 11), Zabaikal ((n =11), Mezen ((n = 12), Tuvinka (n = 11), and Khakass(n = 11). The DNA, extracted with the use of the Dia�tom DNA and Extra Gene DNA Prep (OOO Labori�toriya Izogen, Moscow) kits, was amplified on a 2720Thermal Cycler with a Stock Marks primer kit, andthen electrophoresis of the amplificates was carriedout on an ABI3130 automatic four�capillary geneticanalyzer. Interpretation of graphic images and deter�mination of the animals’ genotypes were carried outon the basis of 17 loci of DNA microsatellites withconsideration of a control sample and results of typingsamples in the 2007–2008 Horse Comparison Test.

In conducting the population genetics analysis, wedetermined the frequency of alleles and number ofalleles at the locus (Na), number of private alleles(Pa), degree of observed heterozygosity (Ho), degreeof expected heterozygosity (He), level of polymor�phism (Ae), as well as genetic similarity coefficients.The data were processed statistically by conventionalmethods with the use of Statistica v. 6.0 software.

RESULTS AND DISCUSSION

On testing the examined horses (n = 1070)it wasestablished that improved and local horse breeds differnoticeably in the presence and frequency of alleles of anumber of microsatellite loci (Tables 1 and 2).Thebroadest spectrum of alleles of microsatellite loci (140across 17 loci) as well as the maximum number of pri�

ANIMAL HUSBANDRY

Study of Polymorphism of Satellite DNA of Improvedand Local Horse Breeds1

V. V. Kalashnikov, L. A. Khrabrova, A. M. Zaitsev, L. V. Kalinkova, and M. A. ZaitsevaAll�Russian Horse Breeding Research Institute, Ryazan oblast, 391105 Russia

e�mail: vniik08@mail.ruReceived May 19, 2010

Abstract—Interbreed differentiation of 12 improved and local horse breeds is studied based on 17 DNA mic�rosatellites. Genetic differences between breeds are established; private alleles are noted in the majority ofbreeds. Genetic distances between breeds are calculated, an analysis of which shows considerable genetic dif�ferences between local and improved breeds.

Keywords: horse breeds, PCR, DNA microsatellites.

DOI: 10.3103/S1068367410060194

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STUDY OF POLYMORPHISM OF SATELLITE DNA 461

vate alleles (11) were found in horses of the Akhal�Teke breed. A slightly smaller number of alleles wererevealed in horses of the local Altai breed (129), ofwhich three localized at two microsatellite loci(ASB17 W, V and AHT5 P) were not found in repre�sentatives of other breeds, which can serve as proof ofthe uniqueness of this ancient breed.

Two private alleles each were noted in three localbreeds: Khakass (ASB17 T and CA425 P), Mezen(HMS1 H and LEX3 R), and Zabaikal (HMS2 F andHMS6 Q) and one each in Buryat (HMS7 P) andTuvinka (ASB23 G) (Table 1). Private alleles were notrevealed only in horses of the Vyatka breed; however,this local breed has a characteristic genetic structure.

On comparing the allele pool of horses of fiveimproved breeds (Table 2), we also noted the presenceof individual specific alleles in representatives of thesebreeds being bred in dozens of countries. It turned outthat the presence of four private alleles at four microsat�ellite loci (AHT4 R, ASB2 U, HMS2 N, and HTG4 R)distinguishes even horses of the Thoroughbred breed.Two characteristic alleles each were noted in horses ofthe Arabian (HMS3 L and LEX3 G), Trakehner(AHT5 Q and ASB23 V), and Standardbred (HMS1 Gand HTG7 L) breeds.

The degree of heterozygosity of the examined pop�ulations on average across the loci varied in the intervalfrom 0.605 (Mezen breed) to 0.776 (Tuvinka breed).

Among the improved breeds, the Arabian (0.610) wasthe most consolidated. On the whole, a sufficientlyhigh degree of heterozygosity, 50–100%, was deter�mined in improved and local horse breeds with respectto all 17 microsatellite loci, which allows using micro�satellite polymorphism for assessing heterozygosity ofboth individual animals and populations.

A comparison of the genetic structure of popula�tions with consideration of polymorphism of 17 mic�rosatellite loci revealed a high level of similarity oflocal Siberian breeds—Altai and Zabaikal, Altai and

Table 1. Characteristics of polymorphism of microsatellite DNA of local horse breeds

LocusAltai Buryat Vyatka Zabaikal Mezen Tuvinka Khakass

Na Pa Na Pa Na Pa Na Pa Na Pa Na Pa Na Pa

AHT4 7 – 5 – 8 – 5 – 6 – 7 – 5 –

AHT5 7 P 4 – 3 – 6 – 6 – 4 – 6 –

ASB2 11 – 5 – 6 – 5 – 6 – 7 – 7 –

ASB17 13 W, V 7 – 6 – 7 – 6 – 6 – 7 T

ASB23 6 – 6 – 3 – 6 – 6 – 8 G 6 –

CA425 8 – 6 – 6 – 6 – 5 – 6 – 6 P

HMS1 6 – 4 – 5 – 4 – 4 H 4 – 4 –

HMS2 7 – 4 – 7 – 4 F 5 – 5 – 4 –

HMS3 7 – 4 – 7 – 4 – 7 – 6 – 6 –

HMS6 6 – 4 – 4 – 4 Q 6 – 4 – 4 –

HMS7 6 – 4 P 5 – 5 – 5 – 5 – 5 –

HTG4 7 – 6 – 8 – 6 – 5 – 5 – 4 –

HTG6 5 – 6 – 5 – 6 – 2 – 5 – 4 –

HTG7 6 – 4 – 5 – 4 – 4 – 4 – 3 –

HTG10 10 – 7 – 5 – 7 – 8 – 7 – 7 –

LEX3 11 – 6 – 4 – 6 – 4 R 7 – 7 –

VHL20 8 – 7 – 3 – 7 – 6 – 8 – 8 –

Total 129 3 91 1 90 0 98 2 92 2 89 1 93 2

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Dendrogram of genetic distances between breeds

ArabianAkhal�Teke

ThoroughbredTrakehner

StandardbredZabaikal

AltaiVyatka

KhakassBuryatMezen

Tuvinka

Dendrogram of genetic distances between breeds.

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KALASHNIKOV et al.

Khakass—as well as improved breeds—Thorough�bred and Trakehner. A pronounced genetic divergenceof two branches formed by improved and local horsebreeds is shown on the dendrogram (figure). Improvedbreeds formed two genetically close subgroups: Ara�bian–Akhal�Teke and Thoroughbred–Trakehner, towhich is joined the distant branch of the Standardbredracing breed.

Thus, a comparative assessment of polymorphismof 17 DNA microsatellite loci of 12 improved and localhorse breeds showed that practically each breed has itsown characteristic genetic structure with the presenceof several private alleles. In this case, the most ancientimproved horse breed, the Akhal�Teke, is character�ized by the highest level of polymorphism. A high levelof genetic similarity of domestic local horse breeds,which form a single cluster, was established. Therevealed genetic characteristics of horses of differentbreeds give additional information for studying theirorigin and can be used in programs for preserving thegene pool of small populations.

REFERENCES

1. Ellegren, H., Johanson, M., Sandberg, K., et al., Clon�ing of Highly Polymorphic Microsatellites in theHorse, Anim. Genet., 1992, vol. 23, pp. 133�142.

2. Binns, M.M., Holmes, N.G., Holiman, A., et al., TheIdentification of Polymorphic Microsatellite Loci inthe Horse and Their Use in Thoroughbred ParentageTesting, Br. Vet. J., 1995, vol. 151, pp. 9–16.

3. Bjornstad, G. and Roed, K.H., Breed Demarcationand Potential for Breed Allocation of Horses Assessedby Microsatellite Marker, Anim. Genet., 2001, vol. 32,pp. 59–65.

4. Bowling, A.T. and Ruyinsky, A., The Genetics of theHorse, Wallington, UK, CABI Publ., 2000.

5. Luis, C., Juras, R., Oot, M.M., et al., Genetic Diversityand Relationships of Portuguese and Other HorseBreeds Based on Protein and Microsatellite Loci Varia�tion, Anim. Genet., 2007, vol. 38, pp. 20–27.

6. Konskie porody Srednei Azii (Horse Breeds of CentralAsia), Vitt, V.O. (Ed.), Moscow, 1937.

Table 2. Characteristics of polymorphism of microsatellite DNA of improved horse breeds

LocusThoroughbred Arabia Akhal�Teke Trakehner Standardbred

Na Pa Na Pa Na Pa Na Pa Na Pa

AHT4 8 R 8 – 9 F, Q 4 – 5 –

AHT5 6 – 5 – 5 – 6 Q 5 –

ASB2 12 U 12 – 12 G, H 11 – 6 –

ASB17 5 – 10 – 10 – 7 – 10 –

ASB23 6 – 8 – 8 – 7 V 6 –

CA425 6 – 8 – 8 – 3 – 6 –

HMS1 4 – 5 – 5 – 3 – 4 G

HMS2 8 N 8 – 8 – 6 – 7 –

HMS3 7 – 10 L 10 G, J 6 – 6 –

HMS6 6 – 7 – 7 J 6 – 5 –

HMS7 9 – 6 – 7 – 7 – 6 –

HTG4 8 R 5 – 7 – 5 – 5 –

HTG6 8 – 4 – 6 – 6 – 3 –

HTG7 4 – 4 – 5 P, I 3 – 4 L

HTG10 6 – 9 – 8 – 6 – 6 –

LEX3 8 – 11 G 9 – 5 – 6 –

VHL20 8 – 7 – 11 K, S 7 – 7 –

Total 104 4 102 2 140 11 100 2 97 2