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Сельскохозяйственная биология, 2010, № 6, с. 20-25.

УДК 636.2:575.174:577.212

THE ANALYSIS OF 30 MICROSATELLITE MARKERS IN LOCAL CATTLE POPULATIONS

T.Y. Kiselyova¹, B.Y. Podoba², Y.Y. Zabludovskiy², V.P. Terletskiy¹, N.I. Vorobyev¹, J. Kantanen³

An artificial insemination used in commercial cattle breeds has resulted in a loss of genetic diversity. Local cattle breeds may possess unique alleles and their combinations as a result of adaptation to different environmental challenges. Microsatellite markers are widely used for population genetic studies of cattle. 30 microsatellite markers (BM1824, BM2113, BM1818, CSRM60, CSSM66, ETH3, ETH10, ETH152, ETH185, ETH225, HAUT24, HAUT27, HEL1, HEL5, HEL9, HEL13, INRA023, INRA032, INRA035 INRA037, INRA063, ILSTS005, ILSTS006, MM122, INRA005, SPS115, TGLA227, TGLA126, TGLA122, TGLA53) in 238 individuals from 6 local cattle populations (Suksun, Istoben, Yaroslavl, Kholmogory, Grey Ukrainian and Pechora type of Kholmogory) were analyzed. In total, 268 alleles were detected from the 30 loci surveyed. Each locus was characterized according to its length, number of alleles, expected heterozygosity, F_is criterion. Genetic estimation of each population was carried out according to total and mean number of alleles, number of private and effective alleles, observed and expected (unbiased) heterozygosity, F_is criteria and interval confidence (IC). As a result wide polymorphism was revealed both in the loci and the populations studied. All populations had stable genetic structure.

Key words: microsatellite markers, alleles, local breeds, cattle.

 

Intraspecific biodiversity of cattle breeds is the subject of interest in terms of population genetics and preservation of the gene pool, and this is also a source of valuable genotypes for practical breeding. The data about genetic structure of species are important in studying the evolution of farm animals. It should be noted that local populations of cattle retain higher individual variability than commercial breeds. An extensive use of artificial insemination has resulted in loss of several valuable alleles. In experiments with Argentine Creole cattle (a local breed from South America) and American Holstein, it has been shown a higher degree of genetic diversity for loci affecting milk production in local cattle (1). Evaluation of Nordic commercial breeds showed the absence of some alleles of the locus for transferrin (2). In Russia, the ongoing tendency for crowding out of local cattle by commercial breeds can lead to irreversible loss of valuable domestic gene pool formed during a long-term adaptation to local environmental conditions.

Local breeds can carry unique combinations of alleles, which is the important reserve of genetic variability for commercial breeds of domestic animals. These alleles must be detected, conserved and used in selection practice (3-5). In this regard, it is extremely actual to identify genetically unique breeds.

Modern studies of population structure use the most common method for genotyping of alleles of microsatellite loci. Their location is important for searching the candidate genes responsible for commercially valuable quantitative traits (QTLs) and the genes providing manifestation of hereditary diseases (which is used in development of methods for prevention and treatment), because microsatellite alleles can be inherited linked to alleles of such genes. Identification of loci responsible for economically valuable traits is based on an the analysis of length of microsatellite DNA fragments using appropriate statistical programs containing the data about dairy and meat productivity (6, 7). The large number of alleles provides accurate identification of any individual since microsatellites are found almost in all living organisms. Currently, evaluation of animal’s origin upon microsatellite markers is more precise than upon blood groups. That’s why the international practice of sale abroad of breeding animals obligates to confirm their origin by the analysis of microsatellite DNA. There are recommendations by FAO about types of microsatellites used for genetic evaluation of cattle (8).

According to INRA (French National Institute for Agricultural research) database, there are 2402 microsatellites identified in cattle today, including 2,244 mapped ones (microsatellites are present on all 30 pairs of chromosomes). Genetic analysis of  populations involves using packages of computer programs - Arlequin, GDA, Gen-AlEx, GENEPOP, GeneClass, Genetic Studio, MicroSat, PowerMarker, which are widely used in agricultural genetics.

The purpose of this work was to study the polymorphism of 30 microsatellite markers in six local populations of cattle.

Technique. The analysis was performed upon DNA extracted from blood samples of 238 animals from populations the following local breeds: Kholmogory (pedigree farm “Arkhangel’skii”, Kholmogorsky district, Arkhangelsk province), Pechora type of Kholmogory cattle (pedigree farm “Ukhta-97”, Ukhta, Komi Republic), Gray Ukrainian (subsidiary farm of Kiev-Pechersk Lavra, Boryspil district, Ukraine), Istoben (pedigree farm “Istobenskii”, Orichevsky district, Kirov province), Suksun (the farm “Savinskoe”, Suksun district, Perm province) and Yaroslavl (the farm “Savinskoe” and the experimental farm “Tutaevo”, Tutaev district, Yaroslavl province).

30 microsatellite markers were used: BM1824, BM2113, BM1818, CSRM60, CSSM66, ETH3, ETH10, ETH152, ETH185, ETH225, HAUT24, HAUT27, HEL1, HEL5, HEL9, HEL13, INRA023, INRA032, INRA035, INRA037, INRA063, ILSTS005, ILSTS006, MM122, INRA005, SPS115, TGLA227, TGLA126, TGLA122, TGLA53.

DNA was isolated by standard phenol method (9). The resulting DNA and primers were diluted with deionized water to concentrations 10 ng/ul and 10 pmol/ul. Conditions of polymerase chain reaction (PCR) were set according to the database of  Roslin Institute (UK). Microsatellites with sharply differing lengths of alleles were analyzed in pairs. PCR products were separated in 6% polyacrylamide gel. Lengths of microsatellites were measured on the devices ALF Express automated DNK sequencer (“Pharmacia”, Sweden) and MegaBACE™ 500 DNA sequencer (“Amersham Biosciences”, USA). To identify alleles of microsatellite loci, the program ALF Win Fragment Analyser 1.00.36 was used. True length of allele was determined by comparing it with lengths of alleles of marker animals. On the obtained charts, locations of peaks were assessed to determine the length of a microsatellite (bp).

Each locus was evaluated by its length, number of alleles, observed and expected heterozygosity, Fis factor. The polymorphism within a population was assessed by total number of identified alleles, observed and expected heterozygosity (unbiased estimate), the average number of alleles per locus, the number of private and effective alleles, F_is coefficient with allowance for the confidence interval.

Statistical analysis of data was performed using the programs GE-NEPOP, Genetix, Convert, and own approved computer programs developed on the basis of common mathematical models (10, 11).

Results. The analysis of 30 microsatellites located on  the 21st pair of chromosomes (1st - 3rd, 5th, 7th -12th, 14th, 15th – 23rd and 26th) revealed polymorphism in all loci (Table 1).

                                                                                                  

Different microsatellites had unequal maximum number of alleles – from 3 (ILSTS005) to 17 (TGLA122, TGLA53). The microsatellite ILSTS005 was designed for zebu-like cattle which has five alleles (12). Totally, 268 alleles in 30 microsatellite loci were found. Expected heterozigosity was quite high for all microsatellites (0,6122-0,8840) except INRA035 (0,4645) and ILSTS005 (0,3871). The microsatellite INRA035 demonstrated fairly close inbreeding: F_is coefficient (shows the decrease of observed heterozigosity compared with expected one) for INRA035 equaled to 0,509 as the sum for all populations. Fis was negative for only microsatellite (MM12; F_is = -0,014) indicating a weak outbreeding; for other microsatellites, it was weakly positive.

The analysis of Hardy-Weinberg genetic equilibrium using the exact method and the Bonferroni correction revealed significant deviations of genotype frequencies of the microsatellite INRA035 in four populations: Suksun (P < 10^-4), Yaroslavl (P < 10^-4), Kholmogory (P = 3×10^-4) and in the Pechora type of Kholmogory (P = 10^-3), since the critical level of significance of individual tests for 30 loci shall not exceed 0,0017 to be significant at 0,05%. In Istoben and Gray Ukrainian breed populations, P-values were, respectively 0,0051 and 0,0541.

All populations differed in number of identified alleles - from 152 to 194 (Table 2). The mean number of alleles per locus for 30 microsatellites amounted to 8,93. According to the literature, this indicator in German Simmental equals 6,64, in Swedish Holstein  - 6,52 for 25 microsatellite markers included in the abovementioned list (5). In Black-and-White cattle, this parameter was 9,00 in the analysis of 7 microsatellites with maximum number of alleles while the higher diversity in length of microsatellites: TGLA227 - 75-105, ETH225 - 132-160, ETH10 - 207-231, BM1818 - 248-278, BM2113 - 151-155, BM1824 - 176-196 and SPS115 - 234-258 bp (13). In this study, in cases when different breed populations had equal number of microsatellite alleles, lengths of these alleles were distinct. Thus, in Yaroslavl and Gray Ukrainian cattle, five alleles of the microsatellite BM1824 were found, whose lengths were, respectively 179, 181, 183, 189, 191 and 179, 181, 183, 187, 189 bp. In this case, the alleles of 187 and 191 bp length were specific for these breeds: the first one was detected in Gray Ukrainian and wasn’t found in Yaroslavl cattle, the second allele was present in Yaroslavl breed and absent - in Gray Ukrainian.

Unique alleles discovered in only one population are of the great interest. In Suksun population, 7 such alleles were identified, in Istoben - 5, in Yaroslavl - 13, in Kholmogory - 6, in Gray Ukrainian - 12 and in the Pechora type of Kholmogory cattle - 1. Gray Ukrainian and Yaroslavl breeds had almost similar  number of unique alleles. It is possible that during the evolution of such breed, its long-time farming in small populations with forced inbreeding resulted in development of inner mechanisms preventing the restriction of genetic diversity. This fact was indicated by the analysis of inheritance of alleles for B-system blood groups in the genetic fund of Gray Ukrainian cattle the herd of pedigree farm “Polivanovka” (14). In this population, actual number of heterozygotes was 18% while the expected value of 28% , and it has been found the intra-gene recombinations resulted in emergence of new alleles (15, 16).

The number of effective alleles per locus (provides main contribution into total homo- and heterozygosity) in breed populations of cattle varied insignificantly: from 2,86 (Gray Ukrainian) to 3,33 (Suksun) (Table 2). No differences in observed and expected heterozygosity as well as in average number of alleles per locus were detected. Coefficient of inbreeding (F_is) showed the negligible prevalence of heterozygous  alleles (the sum over all loci) in Istoben (-0,0104) and in Yaroslavl (- 0,0163) populations, while the other breeds demonstrated low positive values indicating a slight predominance of homozygous alleles (Suksun - +0,0125, Kholmogory – +0,0180, Pechora type of Kholmogory - +0,0088 and Gray Ukrainian - +0,0040). In all groups of animals, Fis was within the confidence interval (95%), which confirmed the absence of both inbreeding and outbreeding, therefore, the studied populations had stable genetic structure.

Thus, the six studied local breed populations of cattle were analyzed upon 30 microsatellite markers, which has revealed a wide  polymorphism of alleles’ lengths. In all of these populations unique microsatellite alleles have been found. The established levels of  observed and expected heterozygosity, as well as F_is-coefficient indicated a stable genetic structure (no inbreeding or outbreeding were reliably detected). Polymorphic microsatellite markers are widely used in breeding work. In the world practice of cattle farming, there are available commercial kits (DNA tests) for identification the individuals with best tenderness and marbling of meat (http://www.bovigen.com, http://www.idenity.com, http://www.nbcec.org).

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1All-Russia Research and Development Institute of Farm Animal Genetics and Breeding, RAAS,  St.-Petersburg – Pushkin 196600, Russia, e-mail: ktyu@yahoo.com, valeriterl@mail.ru, nikolai_vorobyov@yahoo.com; 2Institute of Animal Breeding and Genetics, UAAS, Kyiv province, Borispil region, Chubinsky settlement 08321, Ukraine,   e-mail: mba@kbs.kiev.ua; 3Agricultural Research Centre of Finland Animal Production Research, FIN-31600 Jokioinen, Finland, e-mail: juha.kantanen@mtt.fi

Поступила в редакцию 6 апреля 2010 года

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