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Background: The parthenogenetic Caucasian rock lizard Darevskia armeniaca, like most other parthenogenetic vertebrate species, originated through interspecific hybridization between the closely related sexual Darevskia mixta and Darevskia valentini. Darevskia armeniaca was shown to consist of one widespread allozyme clone and a few rare ones, but notwithstanding the origin of clonal diversity remains unclear. We conduct genomic analysis of D. armeniaca and its parental sexual species using microsatellite and SNP markers to identify the origin of parthenogenetic clonal lineages., Results: Four microsatellite-containing loci were genotyped for 111 specimens of D. armeniaca, 17 D. valentini, and four D. mixta. For these species, a total of 47 alleles were isolated and sequenced. Analysis of the data revealed 13 genotypes or presumptive clones in parthenogenetic D. armeniaca, including one widespread clone, two apparently geographically restricted clones, and ten rare clones. Comparisons of genotype-specific markers in D. armeniaca with those of its parental species revealed three founder-events including a common and two rare clones. All other clones appeared to have originated via post-formation microsatellite mutations in the course of evolutionary history of D. armeniaca., Conclusion: Our new approach to microsatellite genotyping reveals allele-specific microsatellite and SNP markers for each locus studied. Interspecies comparison of these markers identifies alleles inherited by parthenospecies from parental species, and provides new information on origin and evolution of clonal diversity in D. armeniaca. SNP analyses reveal at least three interspecific origins of D. armeniaca, and microsatellite mutations in these initial clones give rise to new clones. Thus, we first establish multiple origins of D. armeniaca. Our study identifies the most effective molecular markers for elucidating the origins of clonal diversity in other unisexual species that arose via interspecific hybridization.

One of the main questions-in the study of the unisexual (parthenogenetic) species of vertebrates is the determination of their genetic diversity. Nuclear and mitochondrial genetic markers can be used for this purpose. One of the most effective genetic markers is the microsatellite DNA, which mutates at a high rate. Development and characteristics of such markers are necessary in studies of parthenogenetic species. In this work, the analysis of the allele polymorphism of three microsatellite loci was performed for the first time via locus-specific PCR in the populations of parthenogenetic species Darevskia rostombekovi (n = 42) and bisexual parental species D. raddei (n = 6) and D. portschinskii (n = 6). All examined individuals of the parthenogenetic D. rostombekovi were heterozygous. Two to five alleles, depending on the locus, were found in the studied populations of the parthenogenetic species. It was shown that the differences were due to the varying structure of the microsatellite cluster and to single nucleotide substitutions at fixed distances in the DNA regions adjacent to the cluster. The allele structure variations form haplotype markers specific for each allele and inherited from the parental bisexual species. It was determined which alleles of the parthenogenetic species were inherited from the maternal species and which from the paternal species. Characteristics of distribution, frequency of occurrence, and combination of alleles of microsatellite loci, which determine the distinctive features of each D. rostombekovi population were obtained. The data can be used in the future to determine the clonal diversity and possible ways of its formation in the populations of the parthenogenetic species D. rostombekovi.

The study of VNTR-polymorphism and the molecular struc ture of 3'-UTR of the dopamine transporter gene (DAT1/SLC6A3) was performed in hadza and datoga males. It was shown that hadza and datoga differed in allele and genotype frequencies. Allele with 9 repeats in 3'-UTR is more common in hadza as well as homozygous genotype DAT19/9. Allele with 10 repeats is more common in datoga as well as homozygous genotype DAT1 10/10. The molecular structure of the DAT alleles with 3, 8 and 12 repeats was determined for the first time. In addition it was found that DAT1 allele with 11 repeats in datoga significantly differed from previously described ones in other populations in repeats type and arrangement. We suggest that variations of the repeats num ber and type in the 3'-UTR of allelic variants may affect the dopamine transporter gene function.

In the genome of unisexual (parthenogenetic) lizard Darevskia armeniaca, highly variable locus Du 161 (arm) was discovered. Analysis of allelic polymorphism was carried out using locus-specific PCR of the lizard DNA specimens from 13 isolated Armenian populations (N = 138). In the sample examined, a total of 12 Du 161(arm) alleles were identified, and their differences at the level of primary DNA structure were determined. Sequence analysis of the Du 161 (arm) alleles showed that their microsatellite clusters contained repeats of one type (GATA repeats). Allelic Du 161 (arm) variants differed in the number of GATA monomers in microsatellite, point mutations of transition and transversion types, located at fixed distances from microsatellite cluster, and by single nucleotide insertions, as well as by longer insertions located within and outside of the microsatellite cluster. Moreover, point mutations formed different combinations (haplotypes), typical of certain alleles. These combinations can be used for the analysis of the origin and inheritance of these alleles in D. armeniaca, as well as for investigation of their interspecific variation in the representatives of the genus Darevskia.

Experimental data on the molecular structure and variability of microsatellite loci in unisexual and bisexual lizard species of the genus Darevskia were analyzed. The allelic variants of Du281 and Du47 were found to differ in the number of monomers, the structure of microsatellite clusters, and point mutations in these clusters and flanking DNA. Interspecific comparison of alleles of these loci revealed both variable regions in the microsatellite clusters and allele-specific evolutionarily conserved nucleotide groups. In general, the results of comparative structural analysis of allelic variants testify to a high genetic similarity of the unisexual and bisexual lizard species studied and reveals the characteristic features of their interspecies variability.

Using a pair of primers selected for the loci deltau 215, deltau 281, and deltau 323 of Darevskia unisexualis monolocus PCR analysis of orthologous loci was carried out in populations of the related parthenogenetic species D. armeniaca and in populations of bisexual parental species D. valentini and D. mixta. It was shown that the studied loci were polymorphic and represented in populations of D. armeniaca by several allelic variants. We cloned and sequenced PCR amplification products of the allelic variants of deltau 215, deltau 281, and deltau 323 loci. It was found that allelic differences of microsatellite loci were caused by variation in a number of tandem repeats in the microsatellite clusters and point mutations in the flanking regions. Interspecies comparison of the orthologous locus deltau 215 between parthenogenetic species D. armeniaca and parental species showed that two allelic variants of deltau 215 in D. armeniaca were inherited from the parental bisexual species D. mixta and D. valentini. The third allelic variant was not found in parental species and appeared because of mutation processes in genome of parthenospecies. For the first time, the information about the molecular nature of allelic polymorphism of these microsatellite loci of parthenogenetic species D. armeniaca was received in this study.

Populations of parthenogenetic lizards of the genus Darevskia consist of genetically identical animals, and represent a unique model for studying the molecular mechanisms underlying the variability and evolution of hypervariable DNA repeats. As unisexual lineages, parthenogenetic lizards are characterized by some level of genetic diversity at microsatellite loci. We cloned and sequenced a number of (GATA)n microsatellite loci of Darevskia unisexualis. PCR products from these loci were also sequenced and the degree of intraspecific polymorphism was assessed. Among the five (GATA)n loci analysed, two (Du215 and Du281) were polymorphic. Cross-species analysis of Du215 and Du281 indicate that the priming sites at the D. unisexualis loci are conserved in the bisexual parental species, D. raddei and D. valentini. Sequencing the PCR products amplified from Du215 and Du281 and from monomorphic Du323 showed that allelic differences at the polymorphic loci are caused by microsatellite mutations and by point mutations in the flanking regions. The haplotypes identified among the allelic variants of Du281 and among its orthologues in the parental species provide new evidence of the cross-species origin of D. unisexualis. To our knowledge, these data are the first to characterize the nucleotide sequences of allelic variants at microsatellite loci within parthenogenetic vertebrate animals.

Using monolocus PCR analysis with the pairs of primers designed for the Du215 locus of Darevskia unisexualis, allelic polymorphism at the orthologous locus in the populations of the related parthenospecies D. armeniaca was investigated. It was demonstrated that Du215 (arm) locus was polymorphic and in the populations of parthenospecies D. armeniaca (n = 127) represented by at least three allelic variants, differing from each other by the size and composition of microsatellite cluster, and by single nucleotide substitutions in flanking DNA. Unlike the Du215 locus, Du215 (arm) was shown contain not only GATA, but also (GACA) repeats, which were absent in D. unisexualis. Thus, in this study, the data on the molecular nature of allelic polymorphism at one of the microsatellite loci of the parthenospecies D. armeniaca were reported.

The genesis of mini- and microsatellite loci, which is under extensive study in humans and some other bisexual species, have been virtually overlooked in species with clonal mode of reproduction. Earlier, using multilocus DNA fingerprinting, we have examined variability of some mini- and microsatellite DNA markers in parthenogenetic lizards from the genus Darevskia. In particular, mutant (GATA)n-restrictive DNA fragments were found in Darevskia unisexualis. In the present study, we examined intraspecific polymorphism of three cloned loci of D. unisexualis--Du323, Du215, and Du281--containing (GATA)7GAT(GATA)2, GAT(GATA)9, and (GATA)10TA(GATA) microsatellite clusters, respectively. Different levels of intrapopulation and interpopulation variability of these loci were found. Locus Du281 showed the highest polymorphism--six allelic variants (in the sample of 68 DNA specimens). Three alleles were found for locus Du215. The Du325 locus was electrophoretically invariant. The primers chosen for loci Du323, Du215, and Du281 were also used for PCR analysis of homologous loci in two presumptive parental bisexual species, D. valentini and D. nairensis. The PCR products of the corresponding loci of the parental species had approximately the same size (approximately 200 bp) as their counterparts in D. unisexualis, but the polymorphism levels of the paternal, maternal, and hybrid species were shown to be somewhat different. These data on the structure of the D. unisexualis loci provide a possibility to study genetic diversity in the parthenogenetic species D. unisexualis and other related unisexual and bisexual species of this genus, which can provide new information on the origin of parthenogenetic species and on the phylogenetic relationships in the genus Darevskia. These data can also be used for resolving problems of marking the lizard genome, which is still poorly studied.

Mini- and microsatellites, comprising tandemly repeated short nucleotide sequences, are abundant dispersed repetitive elements that are ubiquitous in eukaryotic genomes. In humans and other bisexual species hypervariable mini- and microsatellite loci provide highly informative systems for monitoring of germline and somatic instability. However, little is known about the mechanisms by which these loci mutate in species that lack effective genetic recombination. Here, multilocus DNA fingerprinting was used to study M13 minisatellite and (GATA)n microsatellite instability in the parthenogenetic Caucasian rock lizard Darevskia unisexualis (Lacertidae). DNA fingerprinting of 25 parthenogenetic families, from six isolated populations in Armenia (comprising a total of 84 siblings), using the oligonucleotide (GATA)4 as a hybridization probe, revealed mutant fingerprinting phenotypes in 13 siblings that differed from their mothers in several restriction DNA fragments. In three families (8 siblings), the mutations were present in the germline. Moreover, the mutant fingerprint phenotypes detected in siblings were also present in population DNA samples. No intrafamily variations in DNA fingerprint patterns were observed with the M13 minisatellite probe. Estimates of the mutation rate for (GATA)n microsatellite loci in D. unisexualis showed that it was as high as that seen in some bisexual species, reaching 15% per sibling or 0.95% per microsatellite band. Furthermore, in one case, a somatic (GATA)n microsatellite mutation was observed in an adult lizard. These findings directly demonstrate that mutations in (GATA)n microsatellite loci comprise an important source of genetic variation in parthenogenetic populations of D. unisexualis.

Methods of estimating within- and between-population gene diversity in parthenogenetic species using mini- and microsatellite DNA markers and modified Wright's FST statistic are presented with special reference to model populations of lizards of the genus Darevskia (D. dahli, D. armeniaca, D. unisexualis). We used DNA fingerprinting data for several populations of these species examined earlier. The effects of variation in M13, minisatellite, (GACA)n and (TCC)n microsatellite loci on the formation of within-population gene diversity in parthenogenetic species D. dahli and D. armeniaca were shown to be different. The equality of the realized gene diversity H and its maximum possible value Hmax in two populations of D. dahli (Hmax = 0.032, H = 0.031, P < < 0.0431; Hmax = 0.024, H = 0.027, P = 0.09) and D. armeniaca (Hmax = 0.05, H = 0.053, P = 0.03; Hmax = 0.054, H = 0.055, P = 0.02) suggests that variation in (GACA)n loci substantially contributes to the maintenance of within-population genetic diversity. Analysis of between-population genetic diversity using loci M13, (GACA)n, and (TCC)n showed differentiation of D. dahli populations from northeastern and northwestern Armenia (FST = 0.0272, P = 3 x 10(-13)) and genetic homogeneity of the Armenian and Introduced to the Ukraine populations of D. armeniaca characteristic of one clone (FST = 0, P = 1).

Population and family samples of two morphological forms (mutant and normal with respect to dorsal color) of pathogenetic lizard Darevskia armeniaca were examined by means of DNA fingerprinting using M13 mini- and (GATA)n and (TCC)n microsatellite DNA markers. The morphological forms examined were characterized by clonally inherited, species-specific patterns of the DNA markers, which were different from the species-specific DNA fingerprints of the other parthenogenetic species of the genus Darevskia (D. dahli. D. unisexualis, and D. rostombekovi). The mean index of similarity (S) obtained for a sample of 36 individuals from three isolated populations using three types of DNA markers was 0.966. This was similar to the variability level observed in D. dahli (0.962) (P > 0.05), but higher than that in D. unisexualis (0.950) (P < 0.05) and D. rostombekovi (0.875) (P < 0.01). Inheritance of M13 minisatellite and (TCC)n microsatellite DNA markers in the F1 offspring of parthenogenetic lizards was examined. It was shown that variability and clonal diversity of the fingerprint phenotypes observed in the populations and families of D. armeniaca could be at least partly explained by RFLP mutations in microsatellite repeats.

Variation and clonal diversity in populations of the parthenogenetic rock lizard Darevskia rostombekovi was examined by means of multilocus DNA fingerprinting using mini- and microsatellite DNA markers M13, (GATA)4, and (TCC)50). The animals examined were shown to exhibit a clonally inherited, species-specific pattern of DNA markers (fingerprint profile) that is different from the species-specific patterns of parthenogenetic species D. dahli, D. armeniaca, and D. unisexualis. The mean intraspecific similarity index S was 0.950 (0.003) for a sample of 19 animals from three isolated populations of North Armenia. This significantly differed from the estimate of this parameter for a sample of 21 animals including two individuals from mountainous, relict population from the vicinity of the Sevan Lake, which was equal to 0.875 (0.001). A comparison of DNA fingerprints showed differences between 21 individuals attaining 79 DNA fragments of 1801 mini- and microsatellite markers included in the analysis. The results obtained show that intraspecific variation in D. rostombekovi is higher than that in the previously studied parthenogenetic species D. dahli (S = 0.962) and D. unisexualis (S = 0.950) (P < 0.001). Taking into account that D. rostombekovi is considered monoclonal on the basis of allozyme data, the problem of clonal variability is discussed with regard to the evidence on nuclear DNA markers. It is suggested that the hybrid karyotype of D. rostombekovi, which is more unstable than that of D. dahli and D. unisexualis, generates a series of chromosomal rearrangements (mutations). This may lead to the appearance of a geographically isolated chromosomal race (clone) in the population inhabiting the southeastern coast of the Sevan Lake.

In four isolated populations of parthenogenetic Caucasian rock lizard Lacerta unisexualis, variability of (TCC)n loci was examined using multilocus DNA fingerprinting. Unexpectedly high variability of (TCC)n microsatellites was found in all four populations. The mean similarity index was 0.825, which is higher than similarity estimates obtained for other mini- and microsatellite loci in L. unisexualis and parthenogenetic species L. dahli and L. armeniaca studied earlier. The high variation level of (TCC)n loci was shown to be at least partially associated with the presence of a diverged (TCC)n sequence fraction in the L. unisexualis genome. Mutations at some other genetically unstable (TCC)n loci may cause their structural diversity in populations of L. unisexualis.

Multilocus DNA fingerprinting was used to analyze the genome variation of mini- and microsatellite DNA regions in parthenogenetic Caucasian rock lizard Lacerta unisexualis. The DNA fingerprints obtained with probe M13 were nearly identical in all populations examined (the average similarity index S = 0.992). The fingerprints obtained with probe (GATA)4 varied (S = 0.862). Polymorphic fragments were assumed to correspond to allelic variants of genetically unstable GATA loci. Comparison of the fingerprints of animals from four geographically isolated populations revealed several population-specific GATA microsatellite markers. Based on their distribution among the populations, the corresponding alleles were assumed to originate from a common ancestral allele.

DNA fingerprinting was used to estimate genetic diversity within the endangered Siberian crane (Grus leucogeranus) captive population consisting of several dozens of founders originating from the two wild populations of eastern and western Siberia. Similarity and difference among captive individuals were demonstrated by the unweighted pair-group (UPGMA) clustering procedure. Quantitative characteristics of the eastern and western captive population groups such as average percentage differences (APD) and heterozygosity showed a high extent of genetic variability of 77.9-79.3% and heterozygosity of 0.85-0.72 within each group. Genetic heterogeneity of the captive population structure observed here provides guidelines for management of the species gene pool in captivity. These data also indicate that monitoring of genetic diversity through DNA fingerprinting can facilitate the efforts of Siberian crane management and restoration.