Comparison of ISSR Polymorphism among Cattle Breeds

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1 ISSN , Russian Journal of Genetics, 2011, Vol. 47, No. 2, pp Pleiades Publishing, Inc., Original Russian Text Yu.A. Stolpovsky, M. Ahani Azari, A.N. Evsukov, N.V. Kol, M.N. Ruzina, V.N. Voronkova, G.E. Sulimova, 2011, published in Genetika, 2011, Vol. 47, No. 2, pp ANIMAL GENETICS Comparison of ISSR Polymorphism among Cattle Breeds Yu. A. Stolpovsky a, M. Ahani Azari b, A. N. Evsukov a, N. V. Kol a, M. N. Ruzina a, V. N. Voronkova a, and G. E. Sulimova a a Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia; stolpovsky@vigg.ru b College of Agriculture and Natural Resources, University of Tehran, Faculty of Husbandry, Tehran, Iran Received November 25, 2009 Abstract Polymorphism analysis of DNA fragments flanked by (AG) 9 C and (GA) 9 C inverted dinucleotide microsatellite repeats in 766 animals of 19 cattle breeds and one breeding type revealed 66 fragments, of which 64 were polymorphic. The breeds proved to differ in the frequency and presence or absence of amplified DNA fragments at the genomic level, indicating that ISSR fingerprinting is informative for differentiating the PCR product spectra and cattle breeds. Multilocus ISSR polymorphism analysis identified the group of fragments that can be used as Bos taurus and B. indicus species markers to describe the standards of breeds, their genetic profiles, and breed-specific patterns. Based on ISSR polymorphism, a prototypal gene pool of cattle was constructed and the breeds closest to it were identified. Genetic diversity analysis made it possible to assume that an optimal mean heterozygosity is characteristic of cattle breeds and that deviations from this optimum are indicative of various processes occurring in the population (breed). DOI: /S INTRODUCTION Comparisons of DNA polymorphism in farm animals are used to study their genetic variation, differentiation, characteristics, and phylogeny and to identify the association with phenotypic traits. DNA markers substantially expand the possibilities of population genetic analysis and allow the study of among- and within-breed genetic variations of individual genome regions and the genetic structure of a breed. A broad set of markers and methods is used in such studies. One of the most efficient approaches is to use multilocus DNA markers amplified in the polymerase chain reaction (PCR). This group includes markers whose amplification is carried out with random primers (randomly amplified polymorphic DNA, RAPD); primers that have additional artificial sequences (adapters) (amplified fragment length polymorphism, AFLP); or primers complementary to repetitive elements of the genome, such as microsatellites (inter-simple sequence repeat, ISSR). Such markers all have their advantages and drawbacks and vary in resolution, being suitable for population genetic studies [1 3]. ISSR analysis is highly informative because polymorphism is simultaneously estimated for approximately 40 loci. The primers used to develop ISSR markers are complementary to microsatellite repeats (4 12 repeat units) and have one or two arbitrary nucleotides at one end [4]. Such primers allow amplification of the DNA fragments that are between two closely spaced microsatellite sequences. In this work, we used ISSR analysis with two primers, (AG) 9 C and (GA) 9 C, to study the genetic variation of 19 cattle breeds. We identified species- and breed-specific fragments, constructed species- and breed-specific patterns on their basis, and estimated the genetic distances and phylogenetic relationships of the breeds. MATERIALS AND METHODS We examined 761 (in the case of AG-ISSR markers) or 766 (in the case of GA-ISSR markers) animals of 19 cattle breeds and one breeding type (the Caucasian type of the Brown Schwitz breed from the Republic of Daghestan), which was represented by two populations from the SPK Druzhba (n = 48) and GUP Dylymskoe (n = 56). Of the six breeds of the Russian origin, two were developed in the former Soviet Union (Bestuzhevskaya (n = 54) and Kostromskaya (n = 60)), and the other four were of a more ancient origin (Kalmytskaya (n = 29), Gray Steppe (Ukrainian) (n = 45), Yaroslavskaya (n = 62), and Yakutskaya (n = 30)). DNA samples of the foreign breeds N Dama (n = 30); New Zealand Friesian (n = 29); Holstein-Friesian (n = 25); Norwegian Red (n = 30); French Normande (n = 23) and Montbeliard (n = 29); and Indian (Bos taurus indicus) Hariana (n = 9), Tharparkar (n = 9), and Sahiwal (n = 7) were obtained from the Agricultural University College of Dublin (Ireland). DNA samples of the German Black Pied breed (n = 42) were obtained from the Animal Breeding Research Center (Academy of Agricultural Sciences, Düsseldorf Rostock, Germany). Blood samples of Mongolian Hogor- 189

2 190 STOLPOVSKY et al. ogo (n = 47) and South Gobi (n = 47) cattle were collected during a joint Russian Mongolian expedition. ISSR-PCR analysis was performed with the primers (AG) 9 C and (GA) 9 C, which annealed to the (ТС) n and (СТ) n microsatellites. Amplification included initial denaturation at С for 2 min; a number of cycles of denaturation at 94 С for 30 s, annealing at 55 С for 30 s, and synthesis at 72 С for 2 min; and last synthesis at 72 С for 7 10 min. The amplification cycle number ranged from 35 to 37. The amplification product was fractionated in 2% agarose gel at 120 V for min; a GeneRuler 100-bp DNA ladder plus (Fermentas) was used as a molecular weight marker. After electrophoresis, gels were stained with ethidium bromide and photographed in short-wave UV light. The sizes and numbers of fragments in the ISSR patterns were determined using the Onedscan program. Binary matrices were constructed with 1 and 0 corresponding to the presence or absence of a fragment in a pattern, respectively. To identify the DNA fragments of certain sizes in the PCR products more exactly, we developed a universal scale with a grading by molecular weight. A 10- to 200-bp increment was used depending on the region (minor, average, or major). We identified 38 regions with fixed increments in this manner, which allowed us to assign a fragment to a region on the universal scale with a sufficient accuracy. The universal scale makes it possible to reveal regularities at the fragment distribution and to comparatively study the amongbreed and, then, among-species differences. Statistical analysis of data was carried out using the standard programs Statistica 8.0 [5] and Popgene 1.32 [6]. Genetic distances among within-breed groups were estimated according to Nei [7]. The population genetic parameters included in the analysis were based on published studies [8, 9]. RESULTS AND DISCUSSION Generally, the cattle breeds under study differed in both the presence or absence of particular fragments (loci) and their frequencies. Our analysis of the two types of ISSR markers revealed 66 DNA fragments. The (AG) 9 C primer allowed amplification of 37 PCR products, of which 35 were polymorphic. The (GA) 9 C primer produced 29 PCR products, and all of them were polymorphic. The fragments ranged from 160 to 2500 bp. Four fragments amplified with the (AG) 9 C primer were common for the overwhelming majority of animals: fragment A13 = bp (frequency 0.994), which was not observed for one Normande animal; A24 = bp (1.000); A25 = bp (0.994), which was not observed for one Normande animal; and A33 = bp (1.000). Fragment A20 = bp was also common, occurring in the majority of the populations at a frequency of 1.0 with the exception of Mongolian Hogorogo (frequency 0.854); French Montbeliard and Normande (0.678 and 0.792, respectively); and Indian Sahiwal (0.622), Tharparkar (0.529), and Hariana (0.667) cattle (Table 1). Polymorphism analysis with the (GA) 9 C primer showed that two fragments, G23 = bp and G25 = bp, occurred at a frequency of 100% in all but one population. The only exception was the Normande population, where three animals lacked fragment G23 and one animal lacked fragment G25 (Table 2). The above results inidicate that the above PCR products are markers of the two main cattle groups (Bos taurus and Bos indicus) and that a set of seven fragments (A13, A20, A24, A25, A33, G23, and G25) is typical of cattle. The majority of European breeds significantly differ in the frequency of fragment A20 = bp from Asian breeds, in particular, Indian zebu cattle (Bos indicus). Breed-specific fragments were identified in the multilocus patterns as well as the fragments common for the majority of cattle breeds. For instance, fragment A4 ( bp) was detected for all animals of the Estonian Red breed (frequency 1.000) and a few animals of the Yaroslavskaya breed (0.008). The fragment was not observed for any of the other breeds. Fragment A5 occurred exclusively in Mongolian Hogorogo cattle at a frequency of 0.022, and fragment A7 was detected only in Mongolian Hogorogo and South Gobi cattle at frequencies of and 0.078, respectively. Fragment A34 was observed exclusively for the Hariana breed (0.057). As for the GA-ISSR markers, we observed five breed-specific amplification products, which were found for some cattle breeds and were absent from the others. Thus, we observed fragment G2 (frequency 0.031) only in animals of the Estonian Red breed, G12 (0.032) in Mongolian Hogorogo cattle, G24 (1.000) in Tharparkar cattle, G31 (0.010) in the Bestuzhevskaya breed, and G34 (0.017) in the Normande breed. However, when general polymorphism of the 19 cattle breeds and one breeding type was considered, breed-specific fragments that marked a particular breed in the analysis with the two primers were rather unique. The breeds mostly differed in the frequency of a fragment (1 and 0) or grouped by the absence or presence of a fragment. For instance, analysis with the (GA) 9 C primer revealed four cases when certain PCR products (G15, G19, G26, and G29) were observed only for two breeds. Fragment G29 was found exclusively in the French breeds. It is of interest that fragment G38 occurred in the patterns of all animals from the Mongolian Sough Gobi sample and the two populations of the Caucasian type of the Brown Schwitz breed, while it was not detected for the other 18 breeds (Table 2). The frequency distribution of all fragments in the 19 cattle breeds and one breeding type is shown in Fig. 1. Significant differences in the frequencies of certain

3 Primer (AG) 9 C 1.0 COMPARISON OF ISSR POLYMORPHISM 191 A1 A3 A5 A7 A9 A11 A13 A15 A17 A19 A21 A23 A25 A27 A29 A31 A33 A35 A Primer (GA) 9 C 1.0 G1 G3 G5 G7 G9 G11 G13 G15 G17 G19 G21 G23 G25 G27 G29 G31 G33 G35 G Fig. 1. Allele (fragment) frequency distributions in the 19 cattle breeds and one breeding type. The 95% confidence intervals of allele frequencies are shown with vertical lines. fragments are seen clearly. The fragments can be classified into several main types: species-specific fragments (frequency 1.0), such as fragment A24 ( bp); common or rare fragments, such as A17 ( bp) and A27 ( bp), respectively; and fragments that might result from mutations and

4 192 STOLPOVSKY et al. Table 1. Frequencies of fragments amplified with the (AG) 9 C primer in 19 cattle breeds and one breeding type Fragments Bestuzhevskaya Brown Schwitz (Druzhba) Brown Schwitz (Dylymskoe) Holstein- Friesian Kalmytskaya Kostromskaya Estonian Red South Gobi cattle Montbeliard N'Dama Norwegian Red Normande Sahiwal Gray Steppe cattle Tharparkar Friesian Hariana Hogorogo Black Pied Yakutskaya Yaroslavskaya IFS* Total n = A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A * IFS, individual fragment size.

5 COMPARISON OF ISSR POLYMORPHISM 193 Table 2. Frequencies of fragments amplified with the (GA) 9 C primer in 19 cattle breeds and one breeding type Fragments Bestuzhevskaya Brown Schwitz (Druzhba) Brown Schwitz (Dylymskoe) Holstein- Friesian Kalmytskaya Kostromskaya Estonian Red South Gobi cattle Montbeliard N'Dama Norwegian Red Normande Sahiwal Gray Steppe cattle Tharparkar Friesian Hariana Hogorogo Black Pied Yakutskaya Yaroslavskaya IFS* Total n = G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G * IFS, individual fragment size.

6 194 STOLPOVSKY et al. occurred at a negligibly low frequency, such as fragments G2 ( bp), A7 ( bp), and some others. Each breed had its specific pattern when various combinations of the above fragments were considered. To determine the breed-specific patterns for domesticated species, we proposed to use only the most common fragments (frequency equal of higher than 0.4) based on ISSR-PCR analysis of sheep breeds (for more detail, see [10]. By analogy with our earlier definition, we propose the use of a species-specific pattern, which is characterized by the fragments that are the most common in populations of a domesticated species. Species- and breed-specific patterns include the fragments that occur at a conventional frequency equal or higher than 0.4 in the species or breed, respectively. Thus, the species-specific pattern obtained with the AG marker for the cattle breeds included 14 fragments: A6, A8, A9, A13, A15, A16, A17, A20, A24, A25, A28, A29, A22, and A35. The pattern obtained with the GA marker included eight fragments: G6, G10, G13, G23, G25, G30, G25, and G36 (Fig. 1). Figure 2 shows the genome profiles (all PCR products observed for a breed; shown black and gray) and breed-specific or typical patterns (fragments occurring at a frequency higher than 0.4; shown black) that were obtained with the AG-ISSR markers for the 19 cattle breeds. This result can be used to identify a standard for each breed. It is seen that the number of fragments distinguishing one breed from another is rather low, varying from 0 to 5 for breed-specific patterns, as was observed, respectively, in the pairs Montbeliard Normande and Gray Steppe Gobi cattle with fragments A6, A9, A17, A27, and A37. Differences in genome profiles are greater and involve 2 15 fragments. For instance, the differences corresponding to the limiting values were observed between the Holstein-Friesian and Friesian breeds (A27 and A31) and between N Dama and Norwegian Red cattle (A2, A9 A11, A14, A18, A19, A21 A23, A26, A27, A30, and A38). The population frequencies of the PCR fragments in the 19 cattle breeds and one breeding type were used to analyze their phylogeny. According to the principle of population systems, which was formulated by Yu.P. Altukhov and Yu.G. Rychkov, genetic diversity of modern populations corresponds to a certain ancestral population, whose gene pool may be conventionally termed the prototypal gene pool [11, 12]. It was also proposed that the prototypal gene pool be reconstructed by averaging the gene frequencies over all modern populations examined. Note that, as was believed in the 20th century, domestication of cattle (including zebu) took place in three main centers: Indian (modern India and Pakistan), Mediterranean (Mediterranean shore), and Southwestern Asian (Asia Minor, the Caucasus, and Iran) [13]. While the aurochs origin of domestic cattle was not questioned, there was no consensus as to the place and time of domestication. Modern methods of DNA analysis shared light onto the problem. It was found that there were two domestication centers and two ancestors of modern cattle and that differences between the two ancestors reached the species level. Based on nucleotide sequence analysis of the mtdna D-loop, wild ancestors of the two groups of cattle breeds (Bos taurus and Bos indicus) diverged thousand years ago, long before their domestication (8 10 thousand years ago) [14]. The cattle prototypal gene pool (model genetic profile of the ancestor), which was constructed in this work and included the breeds of the above two cattle species, corresponds to the divergence time of Bos taurus and Bos indicus. Figure 3 shows the polygons obtained on the basis of AG-ISSR polymorphism for the 19 cattle breeds and one breeding type. Among all populations examined, the greatest similarity to the prototypal gene pool was characteristic of the Friesian (0.015), Black Pied (0.015), and Mongolian Hogorogo (0.016) breeds. The result seems justified, corresponding to the known data on the phylogeny of the given breeds. It is commonly accepted that Friesian cattle is the most ancient European dairy cattle and gave origin to the majority of Black Pied-related breeds, including the German Black Pied breed, which preserved the initial gene pool of Dutch cattle. Likewise, Mongolian Hogorogo cattle are considered to be one of the most ancient Asian cattle groups. Among the Russian breeds, the closest similarity to the prototypal gene pool was observed for the Kalmytskaya (0.032), Kostromskaya (0.035), Bestuzhevskaya (0.041), and Yaroslavskaya (0.048) breeds. Based on the breeding history and modern state of the breeds, it is possible to assume that cattle breeds or consolidated groups are away from the prototypal gene pool. Greater distances characterize synthetic or less consolidated groups, such as the Caucasian Brown Schwitz type from Dylymskoe (0.166). To study the genetic relationships among the breeds in more detail, we used cluster analysis, which provides a visual illustration of the genetic relationships (Fig. 4). On a dendrogram based on the genetic distances obtained with the AG-ISSR markers, the 19 cattle breeds and one breeding type formed two large clusters (the largest one including 13 breeds), which included six small clusters of two or three breeds. In the case of four small clusters, the clustering was unquestionable because the corresponding breeds were related either by geographical region or by breeding history. Thus, Montbeliard and Normande are both French breeds; Sahiwal, Tharparkar, and Hariana are Indian breeds (Bos indicus); Kalmytskaya and Gray Steppe cattle are the breeds developed in Russian steppe regions; and Brown Schwitz (the Caucasian type from the Druzhba

7 COMPARISON OF ISSR POLYMORPHISM 195 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 A16 A17 A18 A19 A20 A21 A22 A23 A24 A25 A26 A27 A28 A29 A30 A31 A32 A33 A34 A35 A36 A37 A38 Fragment BestuzhevskayaHolstein- Friesian Estonian KalmytskayaKostromskaya Red Gobi cattl Montbeliard N Dama Norwegian Red Normande Sahiwal Gray Black Steppe cattle Tharparkar Friesian Hariana Hogorogo Pied Frequency equal of higher than 0.4 Frequency lower than 0.4 Generally, black and gray bands form a genetic profile of abreed, and black bands form its breed-specific pattern Fig. 2. Genome profile and breed-specific patterns obtained with the (AG) 9 C primer for the 19 cattle breeds. Yakutskaya Yaroslavskaya Common for cattle

8 196 STOLPOVSKY et al. Friesian Kalmytskaya Black Pied N Dama Bestuzhevskaya Gray Steppe cattle Brown Schwitz (Druzhba) Yaroslavskaya Brown Schwitz (Dylymskoe) Yakutskaya Hariana Prototypal gene pool Holstein- Friesian Tharparkar Kostromskaya Sahiwal Estonian Red Normande Norwegian Red Mongolian Hogorogo Mongolian Gobi cattle Montbeliard Fig. 3. Polygons based on polymorphism of ISSR markers obtained with the (AG) 9 C primer for the 19 cattle breeds and one breeding type. population) and Kostromskaya have a common origin, since the Brown Schwitz breed was involved in the breeding history in both cases. The clustering of Friesian cattle, which is one of the most ancient breeds, with Norwegian Red can be explained by the fact that the Dutch Friesian breed was an ancestor of many European dairy breeds, including those of Norway and the German Black Pied breed, which is close to the cluster of Friesian and Norwegian Red cattle. The last cluster included the Yaroskavskaya and Holstein-Friesian breeds. Two hypotheses were advanced for the origin of the Yaroslavskaya breed. According to one of

9 Primer (AG) 9 C Friesian Norwegian Red Black Pied Montbeliard Normande Holstein-Friesian Yaroslavskaya Yakutskaya N Dama Bestuzhevskaya Sahiwal Tharparkar Hariana Kalmytskaya Gray Steppe cattle Mongolian Hogorogo Brown Schwitz (Druzhba) Kostromskaya Mongolian Gobi cattle Estonian Red Brown Schwitz (Dylymskoe) COMPARISON OF ISSR POLYMORPHISM Distance Fig. 4. Cluster analysis of the 19 cattle breeds and one breeding type. The dendrogram was constructed by UPGMA on the basis of Nei s genetic distances [7]. them, the Yaroslavskaya breed was developed from Russian cattle via folk-breeding and lacked foreign ancestors. The other hypothesis suggests crosses with ancestors of Dutch cattle for the Yaroslavskaya breed. Our result supports the second hypothesis. It is known, however, that the Yaroslavskaya breed, which is among the best Russian dairy breeds, was crossed with Holstein cattle recently. Therefore, the close relationship of the Holstein-Friesian and Yaroslavskaya breeds (D N = 0.09) most likely results from interbreed crosses of the past decade. The same is true for Yakutskaya cattle, because attempts were made to improve the productivity of local Yakut cattle by crosses with the Holstein- Friesian breed, which is the most productive dairy breed in the world. Consider the cluster that was obtained for the breeds from steppe regions and included Kalmytskaya, Gray Steppe, and Mongolian Hogorogo cattle. There is an opinion that the Mongolian, Kalmytskaya, and Yakutskaya breeds belong to the subspecies Bos taurus turano-mongolicus [15]. This is true only to a certain extent according to our results. Further studies are necessary with both Yakut cattle (remaining pure-bred populations) and Mongolian cattle, which is generally heterogeneous and displays a within-breed phenotypic variation. For instance, Mongolian Hogorogo cattle is an isolated population from northwestern Mongolia and was not involved in interbreed crosses. In contrast, South Gobi populations were possibly affected by crosses with the international Brown Schwitz breed or other breeds of a Brown or Straw Pied origin. The following conclusion is possible from our cluster analysis of the 19 cattle breeds and one breeding type according to Nei on the basis of ISSR markers. Genetic similarity of the majority of breeds is not occasional, but most likely reflects both adaptive similarity of the relevant genes (geographical breeding regions) and the breeding history of the breeds. In other words, the genetic structure of the breeds is associated with their genomic characteristics, which was reflected in the clustering. Genetic diversity among the cattle breeds was estimated according to Kimura, Lewontin, and Nei [6 9]; the results are summarized in Tables 3 and 4. With the (AG) 9 C primer, the greatest diversity was observed for the Norwegian Red, Sahiwal, and Montbeliard breeds, and the lowest diversity was found for the Gray Steppe, Estonian Red, and Kalmytskaya breeds. In the case of the (GA) 9 C primer, the greatest diversity was characteristic of the Normande, Norwegian Red, and Black Pied breeds, and the lowest diversity was observed for the Sahiwal and Friesian breeds (according to Nei) or the Sahiwal, Friesian, and Kalmytskaya breeds (according the Shannon) [6]. The breeds were generally similar in genetic diversity at the AG and GA markers and differed only in qualitative estimates of the variation. The Nei and Shannon indices, which were estimated using 66 PCR products amplified with the primers (AG) 9 C (37 PCR products) and (GA) 9 C (29 PCR products), were h = and 0.113, respectively, and I = and , respectively. Nei s diversity index ranged from to for the AG-ISSR markers and from to for

10 198 STOLPOVSKY et al. Table 3. Genetic diversity parameters estimated with the AG-ISSR markers Breed (population) Number of polymorphic loci n a n e h I Proportion of polymorphic loci, % Friesian Kalmytskaya Black Pied N'Dama Bestuzhevskaya Brown Schwitz (Druzhba) Brown Schwitz (Dylymskoe) Holstein-Friesian Kostromskaya Estonian Red Montbeliard Mongolian Gobi cattle Mongolian Hogorogo Norwegian Red Normande Sahiwal Tharparkar Hariana Yakutskaya Yaroslavskaya Gray Steppe cattle Note: Here and in Table 4, n a is the mean number of alleles per locus, n e is the effective mean number of alleles per locus according to Kimura and Crow [6], h is the mean gene diversity per locus according to Nei [6], I is the mean Shannon information index per locus [6]. For more detail, see [8, 9]. the GA-ISSR markers; Shannon s indices were estimated at and , respectively. Gray Steppe cattle was excluded from analysis in this case because of the lack of polymorphism for the GA markers. This was possibly related to the fact that the animals were sampled from an inbred population. It is known that Gray Steppe (Ukrainian) cattle was propagated in isolation for a long time in Russia and Ukraine and has a critical number according to the FAO classification, thus being at the threshold of extinction [16]. Our ISSR data support this fact. Apart from Gray Steppe cattle, the Kalmytskaya and Estonian Red breeds are distinguished by low diversity parameters based on the AG-ISSR markers among the 19 cattle breeds examined. A possible significance of mean heterozygosity for predicting the stability of a population has long been discussed in population genetic studies. It is believed that a population has a certain optimal heterozygosity and that deviations from the optimum may be indicative of degradation of the gene pool [17, 18]. These assumptions are based on analyses of structural gene polymorphism, which was mostly inferred from heterozygosity for electrophoretic variants of proteins and enzymes. Based on our results, we assumed the following values as optimal mean gene diversity per locus (Nei s indice, respectively): for the AG-ISSR markers and for the GA-ISSR markers. A shift toward lower values may testify to the processes related to inbred depression or general degradation of the gene pool, while a shift toward higher values are most likely indicative of a synthetic origin of the population or directional breeding aimed at preserving genetic diversity, for instance, in a population maintained in situ. The greatest number of alleles per locus was observed for Norwegian Red (1.6053), Brown Schwitz (Caucasian type) (1.473), and Montbeliard (1.473) cattle with the (AG) 9 C primer and for the Normande (1.3421) and Norwegian Red (1.2632) breeds with the (GA) 9 C primer. The effective number of alleles estimates the reciprocal of the heterozygosity and is the allele number such that the calculated heterozygosity is equal to the actual heterozygosity provided that the alleles occur at equal frequencies. Based on this gene diversity parameter, all but one (Gray Steppe cattle)

11 COMPARISON OF ISSR POLYMORPHISM 199 Table 4. Genetic diversity parameters (mean per locus) estimated with the GA-ISSR markers Breed (population) Number of polymorphic loci n a n e h I Proportion of polymorphic loci, % Friesian Kalmytskaya Black Pied N'Dama Bestuzhevskaya Brown Schwitz (Druzhba) Brown Schwitz (Dylymskoe) Holstein-Friesian Kostromskaya Estonian Red Montbeliard Mongolian Gobi cattle Mongolian Hogorogo Norwegian Red Normande Sahiwal Tharparkar Hariana Yakutskaya Yaroslavskaya Gray Steppe cattle population in our sample had higher within- and among-population diversity estimates. To summarize, our comparative analysis of the 66 ISSR fragments in the 19 cattle breeds revealed both conserved and variable regions at the genomic level. The fragment spectra obtained with the (GA) 9 C primer were more conserved, while those obtained with the (AG) 9 C primer were more variable. Comparative analysis with a combination or two factors, the presence or absence of particular fragments and the frequencies of ISSR markers, allowed us to identify the genomic profiles and species- and breed-specific patterns for the species Bos taurus and the cattle breeds under study. Our estimates of polymorphism of genomic loci may be used as objective characteristics of the variation, consolidation, and a unique character of the gene pool in farm animals, in particular, cattle breeds. The estimates provide a basis for breed identification and construction of a breed genomic standard, which marks a group of animals with genetically determined biological and morphological characters and traits. Our molecular genetic data may be used as criteria to evaluate the key efficiency parameters of animal breeding, such as the identity, differentiation, consolidation, and stability of a breed. This is of immense importance for certification of new breeds, pure breeding, or preservation of a breed as a source of certain hereditary characteristics. ACKNOWLEDGMENTS This work was supported by the subprogram Gene Pools and Genetic Diversity of the program Biological Diversity of the Presidium of the Russian Academy of Sciences, a grant for Support of Scientific Schools (NSh for I.A. Zakharov-Gezekhus school), and State Contract no of the Ministry of Science of the Russian Federation. We are grateful to E.P. Cunningham (United Kingdom), Tsendsuren Tsedev (Mongolia), V.F. Maksimenko (YaNIIZhK, Russia), and G.S. Karaev (Daghestan Breeding Unit, Russia) for blood specimens of several cattle breeds. REFERENCES 1. Jeffreys, A.J., Wilson, V., and Thein, S.L., Individual- Specific Fingerprints of Human DNA, Nature, 1985, vol. 316, pp Sulimova, G.E., DNA-Markers in Genetic Studies: Types of Markers, Their Properties and the Range of Application, Usp. Sovrem. Biol., 2004, vol. 124, no. 3, pp

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