Genetic Variation and De Novo Mutations in the Parthenogenetic Caucasian Rock Lizard Darevskia unisexualis Tatiana N. Badaeva*, Daria N. Malysheva, Vitaly I. Korchagin, Alexei P. Ryskov Laboratory of Genome Organization, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia Abstract Unisexual all-female lizards of the genus Darevskia that are well adapted to various habitats are known to reproduce normally by true parthenogenesis. Although they consist of unisexual lineages and lack effective genetic recombination, they are characterized by some level of genetic polymorphism. To reveal the mutational contribution to overall genetic variability, the most straightforward and conclusive way is the direct detection of mutation events in pedigree genotyping. Earlier we selected from genomic library of D. unisexualis two polymorphic microsatellite containg loci Du281 and Du215. In this study, these two loci were analyzed to detect possible de novo mutations in 168 parthenogenetic offspring of 49 D. unisexualis mothers and in 147 offspring of 50 D. armeniaca mothers . No mutant alleles were detected in D. armeniaca offspring at both loci, and in D. unisexualis offspring at the Du215 locus. There were a total of seven mutational events in the germ lines of four of the 49 D. unisexualis mothers at the Du281 locus, yielding the mutation rate of 0.1428 events per germ line tissue. Sequencing of the mutant alleles has shown that most mutations occur via deletion or insertion of single microsatellite repeat being identical in all offspring of the family. This indicates that such mutations emerge at the early stages of embryogenesis. In this study we characterized single highly unstable (GATA)n containing locus in parthenogenetic lizard species D. unisexualis. Besides, we characterized various types of mutant alleles of this locus found in the D. unisexualis offspring of the first generation. Our data has shown that microsatellite mutations at highly unstable loci can make a significant contribution to population variability of parthenogenetic lizards. Citation: Badaeva TN, Malysheva DN, Korchagin VI, Ryskov AP (2008) Genetic Variation and De Novo Mutations in the Parthenogenetic Caucasian Rock Lizard Darevskia unisexualis. PLoS ONE 3(7): e2730. doi:10.1371/journal.pone.0002730 Editor: Hans Ellegren, University of Uppsala, Sweden Received March 31, 2008; Accepted June 22, 2008; Published July 23, 2008 Copyright: 2008 Badaeva et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was partly supported by the Leading Scientific School and Program of President of Russian Federation for financial support of young scientist 02.120.11.23498 MK-4748.2007.4. and MK-2106.2008.4. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: badaeva_t@mail.ru Introduction Unisexuality in vertebrates has attracted wide attention since it was discovered. In squamate reptiles, unisexuality originates from interspecific hybridization between bisexual species and represents true parthenogenesis [1,2]. They propagate via an aberrant gametogenetic mechanism that inhibits genetic recombination and causes clonal inheritance [1]. Hence the progeny consist of only genetically identical females with clonal inheritance in the next generation. Clonal reproduction and clonal diversity are the two features of unisexual vertebrates that make them attractive as model organisms in such areas as evolutionary ecology, genetics, cellular and molecular biology. Parthenogenetic lizards of the genus Darevskia (formerly Lacerta [3]) were the first reptiles to be identified as unisexual [4]. Seven diploid all-female species are currently known, all from the Caucasus Mountains of Armenia [1,5,6]. Previous studies on these parthenogenetic species revealed some degree of allozyme variation [7–11] and low variability of mitochondrial DNA [12]. However multilocus DNA fingerprinting revealed very high levels of genetic variation in parthenogenetic populations of Darevskia unisexualis, D. armeniaca, D. dahli and D. rostombecovi [13–16]. The possible sources of such variation in parthenogenetic populations may be associated with multiple origins of clones from different pairs of founders, mutations, rare hybridization events, or some level of genetic recombination [8,17–20]. However, the contribu- tion of each of those events to the overall genetic variation remains unknown. The most straightforward and conclusive way to assess the mutational contribution to genetic variation is the direct detection of mutational events from pedigree genotyping [21]. Multilocus DNA fingerprinting with various microsatellite probes detected intrafamily variability of fingerprint patterns in D. unisexualis and D. armeniaca lizards [22,23]. These results imply that unstable loci may exist intheir genomes, but the real nature of such loci and supposed mutations remains obscure. Recently Korchagin et al. (2007) cloned and sequenced a number of microsatellite loci of the parthenogenetic species D. unisexualis. Among several loci analyzed in detail only two, Du281 (GenBank accession number AY 442143) and Du215 (GenBank accession number AY 574928), which contain (GATA)n repeats were polymorphic. However until now there was no information about genetical stability of those loci. In the present work, Du281 and Du215 were tested in a pedigree based analysis to enable the detection of possible de novo mutations in parthenogenetic offspring of D. unisexualis and D. armeniaca lizards. PLoS ONE | www.plosone.org 1 July 2008 | Volume 3 | Issue 7 | e2730