Materials and Methods Reproductively mature females of D. unisexualis and D. armeniaca were collected from natural habitats of western and central Armenia. The animals were maintained in separate enclosures in the laboratory until they began to produce eggs. The eggs were incubated under laboratory conditions. Genomic DNA was extracted from blood by standard phenol-chloroform extraction and resuspended in TE_buf- fer of pH 8.0. The loci Du281 and Du215 were amplified with the previously described primers (Du281: 59TTGCTAATCTGAA- TAACTG39, 59TCCTGCTGAGAAAGACCA39; Du215: 59CAA- CTAGCAGTAGCTCTCCAGA39, 59CCAGACAGGCCCCAA- CTT39) [24]. PCR reaction mix (20 ml) contained 20–40 ng genomic DNA, 16PCR buffer (Dialat), 2 mM MgCl2, dNTP 0.25 mM each, and 0.625 units of Taq-polymerase (Dialat). Amplification conditions were: 94C for 3 min and then 40 cycles of 94C 1 min, 50C for 40 s, 72C for 40 s, followed by 72C for 5 min. The products, averaging about 200 bp in size, were separated by electrophoresis on a 8% native polyacrylamide gel (PAAG) and visualized on a ultraviolet light table following ethidium bromide staining. Amplified fragments were excised from the PAAG, purified and cloned into pMos blue vectors following standard procedures (pMos blueBlunt ended Cloning kit RPN 5110, Amersham Biosciences). The clones were amplified in MOSBlue competent cells grown at 37uC, and sequenced. The PCR products were cloned and sequenced using the chain termination reaction with ABI PRISMH BigDyeTM Terminator v. 3.1 on an ABI PRISM 3100-Avant Genetic Analyzer. The sequences of the allelic variants of the PCR products were compared using the MegAlign program (DNASTAR). All animal procedures were carried out according to ethical principles and scientific standards of the ethical committee of Moscow State University. Results In total DNA samples of 217 lizards (49 mothers and 168 offspring ) of D. unisexualis and 197 lizards (50 mothers and 147 offspring) of D. armeniaca were screened by locus-specific PCR. Mutant alleles were detected as changes in the electrophoretic mobility of PCR amplification products obtained from mother and their offspring. No mutant alleles were detected in D. armeniaca offspring at both Du281 and Du215 loci, and in D. unisexualis offspring at Du215 locus. Figure 1 shows typical example of families where no intrafamily variation of PCR products was revealed. At the same time, 15 mutant alleles among offspring of four D. unisexualis mothers were found at the Du281 locus (Fig. 2). These data show that the Du281 locus of D. unisexualis is highly mutable, with an estimated mutation rate of 0.1428 events per germ line tissue. To analyze the molecular structure of mutant alleles, we cloned and sequenced PCR products of the Du 281 locus from maternal and mutant offspring of D. unisexualis. Comparison of nucleotide sequences of mothers and their offspring revealed mutations only in (GATA)n microsatellite clusters, while no mutations were found in the flanking regions (Table 1). The haplotypes (T-A-T and C-G- C), formed by fixed point mutations in the flanking regions of microsatellite cluster, and specific for allelic variants of Du281 [24] were used to mark maternal and corresponding offspring alleles. In family 1, consisting of the mother and one offspring, the deletion of one GATA monomer in microsatellite cluster was found in both offspring alleles. In family 2, consisting of the mother and five offspring, only one offspring allele marked by haplotype C-G-C was mutant with the deletion of GATATA in the microsatellite cluster of all offspring. In family 3, consisting of the mother and two offspring, an insertion of one GATA monomer was found in the microsatellite cluster of both offspring alleles. Family 4, Mutations in Unisexual Lizard Figure 1. Examples of families where where no intrafamily variation of PCR products was revealed. a – Du281 locus, D. armeniaca family; b – 215 locus, D. armeniaca family; c – 215 locus, D. unisexualis family. Maternal DNAs are marked by M, offspring DNAs are shown by arrows and numbered in each family. doi:10.1371/journal.pone.0002730.g001 consisting of the mother and four offspring, represent a more complicated case. While no mutations were observed among offspring alleles marked by haplotype T-A-T, different pattern of mutation was found among offspring alleles marked by haplotype C-G-C. In three offspring the mutatnt alleles revealed a deletion of one GATA monomer, but in another offspring a GATATA sequence was lost in the microsatellite cluster. In summary, single repeat unit changes dominated (in microsatellite clusters of all offspring of the 1st and 3rd family and offspring 1, 2 and 4 of the 4th family). In two families (all offspring of family 2 and offspring 2 of family 4) maternal and offspring microsatellite differed from each other by the deletion of GATATA imperfect monomer. Mutation may occur in both (1st and 3rd families) or only in one allele (2nd, 4th and 5th family). In three out of four families the patterns of mutations were similar in all offspring. The offspring of family 4 showed different pattern of mutations: in most individuals the mutant allele arose as a result of the loss of one microsatellite monomer, while in another it was the loss of imperfect monomer GATATA. PLoS ONE | www.plosone.org 2 July 2008 | Volume 3 | Issue 7 | e2730