BMC Genomics 2007, 8:427 http://www.biomedcentral.com/1471-2164/8/427 their sequences also spanned the whole reads, suggesting they occur in the genome as longer contiguous arrays. FISH experiments using labeled oligonucleotide probes confirmed this presumption, revealing the presence of both sequences at the termini of all pea chromosomes (Fig. 3D–E). To check for specificity of this assay, control experiments were run using probe sequences differing in a single-base substitution within the repeat monomer ((TTACG)n and (TTTACG)n). These sequences were not present in the 454 reads and corresponding probes gave no hybridization signals on mitotic chromosomes (data not shown). Comparative analysis of the repeat composition of pea, soybean and M. truncatula genomes In addition to the identification of the major components of pea repetitive DNA, we were interested in using the 454 sequence data for comparing the pea genome composi- tion to that of related legume species. We primarily focused on soybean (Glycine max), as it was the only spe- cies for which whole genome shotgun reads produced by the same technology were available. The soybean 454 data included 718,589 reads with an average length of 109 nucleotides [19]. To investigate which sequences are shared between these two genomes we identified and ana- lyzed pea 454 reads producing significant similarity hits (E-value <= 1e-10) in BLAST searches against a database of the soybean 454 reads. A total of 5,482 pea reads (1.7%) matched soybean sequences; in the soybean dataset, 7,209 reads (1.0%) gave significant hits with pea. The composition of these sequences is summarized in Fig. 5A–B. The largest fraction of the matching reads belonged to rDNA sequences, which also displayed the highest sequence similarity between the species (reflected by high BLAST bit scores on Fig. 5B). Ogre elements, representing the most abundant repeats in the pea genome, were also identified in soybean but in much smaller numbers. On the other hand, the two species were found to share rela- tively large population of SIRE1 sequences. Interestingly, the other abundant families of pea Ty1/copia elements were absent or gave only a few hits in the soybean genome. In order to compare genome composition of pea and soy- bean with the legume model species Medicago truncatula, we employed the same strategy as above, except for using a set of 197,570 M. truncatula BAC-end sequences instead of 454 data. We found that pea genome contains consid- erably more repeats similar to M. truncatula sequences than soybean. A total of 15,510 (4.9%) pea reads gave sig- nificant matches with M. truncatula and included all major pea repeat families (Fig. 5D). However, there were differences in genomic proportion and sequence conser- vation of individual repeats between pea and M. truncat- ula genomes. For example, abundant pea Ogre sequences were also frequently found in M. truncatula but their sequence similarity was relatively low, whereas Ty1/copia elements (represented mostly by Ps-copia-1/751 family) produced much fewer hits but displayed higher average sequence similarity (Fig. 5D). Contrary to pea, soybean repeats were only poorly represented in M. truncatula. About 2.1% of soybean 454 reads matched M. truncatula sequences, however, except for rDNA (0.25%) no major repeat family was found to be shared by these two species (Fig. 5C). In summary, these results indicate similarity in repeat composition between pea and M. truncatula genomes, and considerable sequence divergence of most repeat families between these two species and soybean. Discussion The rationale behind adapting 454 sequencing to repeat profiling in complex plant genomes is that it provides effi- cient sequence sampling from a high number of inde- pendent genomic loci. The amount of generated sequence data is large enough to include multiple reads from highly repeated elements, thus allowing evaluation of their abundance and sequence composition. However, required prerequisites for the use of 454 sequencing for repeat quantification and reconstruction are that the tem- plate sampling is random and that the sequencing does not introduce a bias towards certain sequences. In this study, we addressed these questions by comparing repeat copy number estimates obtained using experimental approaches [20-24] with the estimates based on the fre- quencies at which these repeats occur in 454 reads. The values were within a two-fold difference range for most of the repeats, and they never differed more than 2.8-fold. The observed discrepancies can be explained by principal limitations of both analytical approaches. The experimen- tal quantification was based on DNA-DNA hybridiza- tions, which may bias estimates if the probe fragment spans sequence regions differing in genomic abundance. In that case, hybridization signal is primarily determined by only a part of the sequence, but it is considered to be representative for the whole probe in subsequent calcula- tions. This is not the case for the calculations of genome representation based on sequence coverage by 454 reads, which are performed separately for each nucleotide of the sequence in question. On the other hand, sensitivity and specificity of sequence similarity searches employed for this analysis can be partially affected by the algorithm and the similarity threshold values used for the assay. Thus, taking into account these limitations, we consider the experimental and 454 data to be in a good agreement. Reconstruction of the repetitive element sequences from 454 reads represents a difficult task, complicated by the short length of the reads and considerable sequence diver- sity of individual genomic copies of the repeat. A similar problem has been successfully addressed by Li and co- Page 8 of 16 (page number not for citation purposes)