BMC Genomics 2007, 8:427 http://www.biomedcentral.com/1471-2164/8/427 reads. This is mainly due to the occurrence of multiple subfamilies of the repeats in the genome and the presence of poorly conserved repeat regions. On the other hand, highly conserved rDNA repeats could be reconstructed as a single contig and their consensus was in excellent agree- ment with the sequences obtained by conventional clon- ing and sequencing of pea rDNA fragments. Instead of performing direct contig assembly from all 454 reads [19], we preceded the assembly with a clustering step, which resulted in partitioning the read collection into groups of overlapping sequences. In addition to reducing the computational complexity of the assembly step, this approach also allowed the classification of con- tigs based on their cluster of origin. In principle, multiple contigs resulting from the assembly of reads from the same cluster should represent overlapping fragments and sequence variants of the same repeat family. Whereas this was true for many repeat families, there were also clusters including reads from several unrelated repeats. This is likely due to the existence of reads containing parts of two different repeats which act as a bridge to join groups of unrelated sequences during the transitive closure cluster- ing procedure. Such reads can, for example, originate from insertion sites of mobile elements, which are numer- ous in the genome and often located within other repeti- tive sequences. This assumption is supported by our results, where this problem occurred in the largest cluster (CL1), which originated from at least five different fami- lies of retroelements. Although such clusters can be subse- quently broken into smaller sets of overlapping contigs (see Methods), we plan to avoid this problem in the future by developing algorithms for identification of such "hybrid" reads and their elimination from the clustering procedure. Our results have shown that low-depth genome sequenc- ing using massively parallel technology provides suffi- cient sequence data for comprehensive repeat characterization even in a relatively large plant genome. Compared to the only other study on this topic, employ- ing 454 sequencing for repeat analysis in soybean [19], the pea 454 sequences used here provided considerably smaller genome coverage (0.77% vs. 7% in soybean) due to the 4-fold difference in genome size between these spe- cies and the smaller reaction scale used in the pea sequencing. Still, it was possible to characterize repeats constituting 35–48% of the pea genome and including all major classes of repetitive DNA. On the other hand, con- sidering the estimated 75–97% proportion of repeats in the genome [5,6], relatively large fraction of the repeats remained uncharacterized. Reassociation kinetics studies of pea genomic DNA [6] as well as observations from other species [33] indicate that this fraction includes diverged, low-copy remnants of ancient repeats ("fossil- repeats"). Such repeats are below the sensitivity limit of our analysis due to their high sequence variability and low copy numbers. Similar to most higher plants studied so far [34-36], LTR- retrotransposons were found to be the major component of pea repetitive DNA. Ty3/gypsy elements were present in twice as many copies as Ty1/copia and constituted an even larger portion of the genome (24–39%, vs. 5% spanned by Ty1/copia) owing to much longer element sequences. The prevalence of Ty3/gypsy elements over other groups of retroelements was observed in other plant genomes including rice [37] and Vicia sp. [38], and their differential proliferation substantially contributed to the genome size variation among related species [9]. In pea, most of the Ty3/gypsy sequences were classified as Ogre-like retro- transposons, a distinct evolutionary lineage of giant ele- ments occurring in a range of dicot plants including the genera of Leguminosae, Solanaceae, and Salicaceae [26]. Ogre elements were found to play an important role in genome evolution of Vicia, a genus closely related to Pisum. They were differentially amplified in individual species, with the highest abundance in V. pannonica where their recent expansion to 105 copies/1C increased the genome size by more than 50% [8]. Contrary to V. pan- nonica, the Ogre population in pea is not as homogeneous but it occurs as several distinct subfamilies differing in their sequences. This suggests that the evolutionary his- tory of Ogre elements in pea was more complex and included processes of amplification and diversification of the elements. Although they are the most abundant, Ogre elements are probably not the only Ty3/gypsy elements with a significant impact on pea genome evolution. For example, Peabody elements were found to be very con- served in their nucleotide sequences, implying their recent amplification. Compared to Ty3/gypsy elements, Ty1/copia represented a much smaller portion of the genome but occurred in a larger number of different families. Intraspecific heteroge- neity of the Ty1/copia population, resulting from the pres- ence of divergent families, was reported in a number of other species [39]. Interestingly, these families are well conserved across different taxa in spite of their ancient ori- gin before the divergence of monocots and dicots [40]. This is also true for the pea Ty1/copia sequences, which in some cases, show high similarity to elements from phylo- genetically distant species (Additional file 5). A typical example is the most abundant family Ps-copia-1/751 with strong similarity to monocot elements RIRE-1 and BARE; moreover, the high proportion of solo-LTRs derived from Ps-copia-1/751 suggests its long presence in the pea genome. Page 10 of 16 (page number not for citation purposes)