BMC Genomics 2002, 3 http://www.biomedcentral.com/1471-2164/3/15 Planaria are prospective models for genetic variation stud- ies in populations. The planarian species (Planariidae fam- ily) are represented by different strains employing various modes of reproduction, including exclusively sexual, ex- clusively asexual, or both sexual and asexual [3–6]. Strains that reproduce exclusively asexually are genetically differ- ent to strains of the same species able to reproduce both sexually and asexually. For instance, an asexual strain of Dugesia (S) mediterranea exhibits morphological hetero- morphosis in the third pair of chromosomes [4]. Worms belonging to different asexual strains of Dugesia japonica and Girardia (formerly Dugesia) tigrina are triploid or mix- oploid, while sexual strains are diploid [5,6]. Asexual strains are represented by independent populations that propagate only by fission. Recently, we supplemented the well-known SSH# (sup- pression subtractive hybridization) technology [7,8] with the Mirror Orientation Selection method (MOS) for elim- inating background clones from SSH-generated libraries [9]. This innovative procedure allowed the application of these techniques in the subtraction of large eukaryotic ge- nomes, including those of freshwater planarian (haploid genome size of G. tigrina is 1.4 × 109 bp [10]). Using the SSH-MOS combination, genomes of two different strains of G. tigrina were compared. Novel virus-like element with unusual features was identified in one planarian strain. We characterized the genome structure of the novel ele- ment and analyzed its distribution in the planarian body. Results Verification of the model Two planarian strains were used for comparison. One was an asexual strain maintained as laboratory culture in Mos- cow (Russia) for over 20 years. Worms belonging to this strain have been successfully used for various experi- ments, including gene discovery [11–13]. The second strain, collected in Russia near Moscow, repre- sented sexual planaria capable of both sexual and asexual reproduction. The specimens were assigned to G. tigrina on the basis of their external appearance [5,14,15]. This strain is morphologically similar to the sexual strain col- lected previously in Spain [5] and like the Spanish ani- mals, differs from asexual planaria in their size and details of coloration. Unlike asexual animals, mature planaria of the sexual strain possess well-distinguished reproductive organs and lay fertile cocoons. To verify the genetic propinquity of the sexual and asexual planarian strains, we analyzed mariner element sequences from the asexual and the sexual specimens. Several DNA sequences similar to the mariner element have been re- ported in G. tigrina [10,16]. There are about 7000 copies of this element in the genome, which appear to code for an active transposase [10]. None of these mariner ele- ments are present in the genomes of other turbellarians, including the species closest to G. tigrina [10]. With the aid of PCR using specific primers for the planarian mari- ner element, we isolated and sequenced several clones from sexual and asexual planaria. All clones displayed 98– 99% identity at the nucleotide level, and the differences between sequences of various strains did not exceed the diversity within one strain. Based on these data, we conclude that the two planarian strains represent a valid model system for studying genetic differences between planarian populations, and can be used for the subtractive hybridization of genomes. Cloning of PEVE Two subtracted libraries were generated: the first enriched with asexual strain-specific DNA fragments (AS library), and the second with sexual strain-specific DNA (SS li- brary). Differential screening revealed 30% and 60% strain-specific clones in SS and AS libraries, respectively. Sequence analyses of selected AS-specific clones demon- strated that about 50% contained open reading frames (ORFs) with codon usage unusual for G. tigrina [17]. Some of these AS-specific ORFs encode polypeptides sim- ilar to viral proteins. When used as probes in Southern blot hybridization with undigested sexual and asexual planarian DNA, these clones stained two relatively low molecular weight DNA bands in asexual specimens only (Figure 1). The intensity of hybridization signals was significantly higher (more then 100-fold) than that for the unique genomic sequence (data not shown). Our results imply that most AS-specific clones are linked to an amplified extrachromosomal virus-like element des- ignated PEVE (Planarian Extrachromosomal Virus-like El- ement). Using PCR with combinations of primers designed from these PEVE sequences and the genome- walking approach, several overlapping fragments consti- tuting the 7.6 kb contig were isolated. PEVE genome organization PCR analyses with PEVE-specific primers showed that the putative viral genome comprises two unique regions flanked by inverted repeats, specifically, Us (1911 bp, GenBank Accession No: AF364089) and Ul regions. Two types of Ul region were identified that displayed some se- quence difference: Ul1 (5699 bp, GenBank Accession No: AF364090) and Ul2 (5695 bp, GenBank Accession No: AF364091). A schematic diagram of the regions with pre- dicted ORFs is presented in Figure 2A. PCR analyses with different combinations of primers specific for PEVE re- Page 2 of 9 (page number not for citation purposes)