BMC Genomics 2007, 8:427 http://www.biomedcentral.com/1471-2164/8/427 This is especially obvious in higher plants, where repeti- tive sequences comprise up to 97% of nuclear DNA [5,6] and contribute significantly to the extraordinary genome size variation observed between different taxa [7-9]. How- ever, the presence of numerous and sequentially diverse families of repetitive elements make their analysis a chal- lenging task. Thus, the most widely used approaches to study the contribution of repetitive DNA to genome evo- lution are based on isolation and characterization of only a single or a small group of elements. These approaches have been valuable in following the fate of various repeats in a wide range of species [10-12]. However, they do not allow for the global comparative analysis of repeat pro- files required for elucidating evolutionary trends on the whole genome level. The demand for a comprehensive repeat analysis prompted the development of a DNA microarray-based assay to study the occurrence of hun- dreds of repeats in twenty plant genomes [13]. Although successful, the microarray-based approach still suffered from several limitations including the need for a priori knowledge of the repeat sequences, the limited capacity of the array, and especially the inability to discover novel repeats for which there were no probes on the array. The requirement for simultaneous determination of sequence composition and abundance of hundreds of repeat families is best fulfilled by analyzing the complete genome sequence; however, such data is available for only a limited number of model species. Alternatively, low- depth shotgun genomic sequencing can be used to survey the most abundant repeats, as was demonstrated for Gos- sypium species [9]. However, performing this type of sur- vey using conventional approaches employing Sanger sequencing is still labor-intensive and requires considera- ble resources. The recent introduction of a massively-par- allel pyrosequencing technology developed by 454 Life Sciences ("454-sequencing") has opened new possibilities for high-throughput genome analysis [14]. This approach allows parallel sequencing of hundreds of thousands of individual templates immobilized on microbeads, thus producing megabases of sequence data in a single run. It has been successfully applied to the sequencing of micro- bial genomes [15], the re-sequencing of mammalian genomes [16], and for transcript profiling [17]. Due to rel- atively short sequence read lengths (~100 nucleotides on average, or ~250 nucleotides with the improved version of the system), the technology is not yet suitable for de novo sequencing of complex genomes. However, it has a great potential for profiling repetitive sequences in these genomes, as the amount of produced sequence data is suf- ficient to get a representative overview of the most abun- dant genomic repeats. For example, a total of 30 Mb determined in a single sequencing run represents only 0.01-fold coverage of a hypothetical 3,000 Mb genome (this is about the haploid genome size of maize or cotton [18]), but provides 10-fold coverage of repeats occurring in the genome in 1,000 copies. The sum of 30 Mb is rep- resented by a set of 300,000 sequence reads which are ran- domly generated from various genomic loci. Theoretically, they should contain fragments of a given repeat randomly sampled from its individual copies, and the frequency of these fragments in the sequence reads should be proportional to the genomic abundance of the repeat. Therefore, this amount of sequence data should be sufficient to reliably detect abundant (at least 500–1000 copies/1C) genomic repeats, and eventually reconstruct their consensus sequences by assembling the reads derived from their individual copies. Recently, this strat- egy has been successfully applied to repeat analysis in the 1,115 Mb genome of soybean [19]. Based on the theoretical considerations described above, we attempted to adapt parallel sequencing technology for the genome-wide characterization of repetitive elements in garden pea (Pisum sativum L.) and for comparative anal- ysis of its repeat composition with other species. In addi- tion to being a classical model for genetic studies, pea is also one of the model species used in our and other labo- ratories for studying the impact of repetitive DNA on leg- ume plant genomes. Consequently, a set of well- characterized pea repetitive elements is available [20-24] which could serve as a control in evaluating the accuracy of the developed assays. Pea has a genome of 4,300 Mb/ 1C [18], which is about 10-fold larger than the genome size of rice or the model legume Medicago truncatula, and about 4-fold larger than the soybean genome. Compared to these species, it is rich in repetitive DNA, which was estimated to comprise 75–97% of its nuclear DNA [5,6]. Our initial experiments were aimed at evaluating the rep- resentation of known repeats in 454 sequence reads. Then we focused on the reconstruction of longer segments of repetitive sequences from multiple overlapping sequence reads, which provided a basis for their further characteri- zation. These data were used to determine the genomic abundance and variability of the major repeat families present in the pea genome. Finally, we used the pea sequence data together with available soybean and M. truncatula sequences to perform comparative analysis of the repeat composition and abundance in these three leg- ume species. Results A single 454 sequencing reaction with pea nuclear DNA resulted in 319,402 usable reads with an average length of 104 nucleotides, yielding a total of 33.3 Mb of sequence data. This is equivalent to 0.77%, or 1/129, of the haploid pea genome (1C = 4,300 Mb). Thus, in theory, repeats occurring at 1,000 copies or greater in the pea genome should be well represented in these sequences, as they Page 2 of 16 (page number not for citation purposes)