BMC Genomics 2007, 8:427 http://www.biomedcentral.com/1471-2164/8/427 domains and a phylogenetic tree was calculated by neigh- bor-joining method (bootstrap values calculated from 1000 replicates) using the ClustalX program [54]. Reverse transcriptase sequences used for the analysis were extracted mostly from plant retrotransposon sequences downloaded from RepBase [55,56]. Graphical representa- tion of the tree was drawn and edited using ATV Forester [57], TreeEdit [58] and Jalview programs [59]. Copy number (CN) estimation based on RT domains was calcu- lated using formula CN = RDrt × 129 × L1/L2, where RDrt is the average read depth within the reverse transcriptase domain, L1 is the length of the reverse transcriptase domain in particular contig and L2 is the length of the full size domain. Contigs bearing only partial reverse tran- scriptase domains and therefore missing in the phyloge- netic tree were assigned to appropriate retrotransposon group based on their best Blast hits to elements shown in the tree (Additional files 4 &5). An alternative approach for copy number estimation of Ogre elements was based on analyzing contigs including the Ogre-specific PBS sequence complementary to tRNAArg [26]. Contig assemblies (ACE files produced by cap3 assembler) were visualized using clview [25] and average read depth over the PBS site was calculated for each contig. The contig sequences were compared using dotter and grouped into three subfamilies according to similarity of the sequences surrounding the PBS. The number of PBS-containing reads was summed for each subfamily and used to calculate its genomic copy number. Microsatellite and telomeric repeats were identified directly in a set of individual 454 reads instead of in the contigs. The reads separated with runs of "N" (to avoid fusing adjacent sequences) were concatenated into a sin- gle sequence and analyzed using Tandem Repeats Finder [60]. The results were sorted and evaluated using TRAP [61]. Comparative analysis of the repeat composition in pea, soybean (Glycine max) and barrel medic (Medicago trunca- tula) was performed using our set of 319,402 pea 454 reads, a set of recently published 718,589 soybean 454 reads [19], and 197,570 M. truncatula BAC-end sequences. The soybean and M. truncatula sequences were down- loaded from the NCBI Trace Archive [48] and from TIGR [62], respectively. Sequence similarities were identified by blastn searches between 454 reads from pea and soybean, and between either pea or soybean 454 reads and M. trun- catula BAC-end sequences. The 454 reads producing blastn hits with E-value of 1e-10 or lower to sequences from the other species were considered to include sequences shared between these genomes. For each of these reads, the number of hits to other genome sequences and average blastn bit score of these hits were determined and plotted using Mgraph (created by Louis Gonzalez and Christine Deroo, available from [63]). Grouping the reads according to the repeat type was done by similarity searches to known sequences performed as described above. Fluorescence in situ hybridization (FISH) Hybridization probes for potential satellite repeats were prepared by PCR amplification from pea genomic DNA using primer pairs designed according to the contig sequences including tandem subrepeats (Table 1 and Additional file 6). The PCR was performed in 30 μl of reaction mix (1× PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 μM primers, 5 U of Taq polymerase (Promega), 0.2–20 ng of template DNA) for 25 cycles of 1 min at 94°C, 1 min at 50°C and 3 min at 72°C, preceded by initial denaturation (3 min at 94°C) and followed by final extension step (10 min at 72°C). Reaction products were resolved on agarose gel electrophoresis and bands correspoding to the repeat monomers or short multimers (< 800 bp) were excised from ladders of the amplified fragments. The excised DNA was purified and used as a template in PCR labeling by biotin-dUTP as described [20]. 5' biotin-labeled oligonucleotide probes for alterna- tive telomeric repeats (CCTAA)8, (CCTAAA)7 and controls (CGTAA)8, (CGTAAA)7-CG were purchased from Bioneer Europe. FISH experiments were carried out on chromo- some squash preparations [64] obtained from root tip meristems synchronized as described [65]. The chromo- some preparations were aged for 5–60 days and post-fixed in 4% formaldehyde/2 × SSC for 10 min at 25°C. The hybridization was performed as described [64] with the following modifications. Probe concentration in the hybridization mix was 0.2 μM for oligonucleotide and 0.5 ng/μl for PCR-amplified fragments. Formamide concen- trations in hybridization and washing solutions and incu- bation temperatures were adjusted according to the probe sequences to achieve 90% stringency for the oligonucle- otide probes (Tm calculated using Exiqon Tm prediction 1.1 software [66]) and 80% stringency for the satellite repeat probes. Stringency for both types of probes was cal- culated according to [64]. Biotin-labeled probes were detected with streptavidin-Alexa Fluor 568 (Invitrogen) and individual chromosome types were distinguished by co-hybridization with Alexa Fluor 488-labeled PisTR-B probe, producing characteristic patterns on each pea chro- mosome [20]. Chromosomes were counterstained with 4,6-diamino-2-phenylindole (DAPI) and observed using a Nikon Eclipse-600 epifluorescence microscope equipped with a CCD camera. The signals were collected using appropriate filter sets and LUCIA software (Labora- tory Imaging). Page 13 of 16 (page number not for citation purposes)