D. N. Malysheva

Instability of (GATA) n microsatellite loci in the parthenogenetic Caucasian rock lizard Darevskia unisexualis (Lacertidae)

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.

Analysis of genetic variation in unisexual and bisexual lizard species of the genus Leiolepis from Southeast Asia

Using multilocus DNA fingerprinting with microsatellite probes (CAC)5, (GACA)4, (GGCA)4, and (GATA)4, intraspecific variation of the Southeast Asian lizards belonging to the genus Leiolepis (bisexual species Leiolepis reevesii and triploid parthenogenetic species Leiolepis guentherpetersi) was first examined. The L. guentherpetersi lizards were characterized by monophyletic DNA fingerprint profiles for the loci detected by the (GACA)4, (GGCA)4, and (CAC)5 probes, in terms of intrapopulation similarity index constituting S = 0.96. This was different from the individual-specific profiles of the lizards from bisexual, presumably parental species, L. reevesii (S = 0.6; P < 0.001). Genetic homogeneity of triploid L. guentherpetersi lizards at the loci examined serves as one of the arguments for the parthenogenetic nature of this species. Genetic variability of triploid parthenogenetic species L. guentherpetersi appeared to be comparable with that reported earlier for the Caucasian rock lizards of the genus Darevskia, namely, D. dahli, D. armeniaca, and D. unisexualis (P > 0.05). The results of DNA fingerprinting analysis of the same L. guentherpetersi samples with the (GATA)4 hybridization probe were unexpected. Variability of parthenogenetic species L. guentherpetersi at the (GATA)n markers was remarkably higher than that at other DNA markers (S = 0.35; P = 3.08 × 10−11), being comparable to the variation of the (GATA)n DNA markers in bisexual species L. reevesii (P = 0.74). The reasons for high polymorphism of the (GATA)n-containing loci in L. guentherpetersi still remain unclear. This polymorhism is probably associated with high instability of the loci, which can be revealed by means of family analysis of parthenogenetic offspring.

Nucleotide sequences of the microsatellite locus Du215 (arm) allelic variants in the parthenospecies Darevskia armeniaca (Lacertidae)

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.

Variation of Mini- and Microsatellite DNA Repeats in Parthenogenetic Lizard Darevskia armeniaca as Revealed by DNA Fingerprinting Analysis

Population and family samples of two morphological forms (mutant and normal with respect to dorsal color) of parthenogenetic lizard species Darevskia armeniaca were examined by means of DNA fingerprinting using M13 mini- and (GACA)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 those 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.

Molecular structure of allelic variants of microsatellite loci Du281 and Du47 in unisexual and bisexual lizard species 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.

Quantitative Assessment of Gene Diversity and Between-Population Differentiation of Parthenogenetic Lizard of the Genus DarevskiaUsing Mini- and Microsatellite DNA Markers

Methods of estimating within- and between-population gene diversity in parthenogenetic species using mini- and microsatellite DNA markers and modified Wrights 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 Hand 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 × 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).

Molecular and genetic characterization of allelic variants of microsatellite loci in parthenogenetic Caucasian rock lizards Darevskia armeniaca (Lacertidae)

Using a pair of primers selected for the loci Du215, Du281, and Du323 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 Du215, Du281, and Du323 loci. It was found that allelic differences in microsatellite loci were caused by variations in the number of tandem repeats in the microsatellite clusters and point mutations in the flanking regions. An interspecies comparison of the orthologous locus Du215 between parthenogenetic species D. armeniaca and parental species showed that two allelic variants of Du215 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 due to 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.

Molecular nature of allelic polymorphism of highly variable microsatellite locus Du161(arm) in unisexual lizard Darevskia armeniaca (lacertidae)

In the genome of unisexual (parthenogenetic) lizard Darevskia armeniaca, highly variable locus Du161 (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 Du161(arm) alleles were identified, and their differences at the level of primary DNA structure were determined. Sequence analysis of the Du161(arm) alleles showed that their microsatellite clusters contained repeats of one type (GATA repeats). Allelic Du161(arm) variants differed in the number of GATA monomers in microsatellite, point mutations of transition and transversion types, located at fixed distances from micro-satellite 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.