BMC Genomics 2006, 7:245 http://www.biomedcentral.com/1471-2164/7/245 repeat motifs of 1–6 bp. We also explored the existence of 7–10 bp motifs, which represent the transition between micro and minisatellites; little is known about these. A total of 84000 available EST sequences were downloaded from the Phytophthora Functional Genomics Database [31] for P. infestans, and 19276 and 16066 predicted and annotated transcripts were downloaded from the Depart- ment of Energy's Joint Genome Institute [32]. The EST set was masked for repetitive sequences, obtained from Rep- Base ([33]; which does not include SSR repeats), assem- bled and revised manually to generate consensus sequences. We used consensus EST sequences because they have the built-in advantage of eliminating redundant SSR counts, allowing us to make more precise estimates of SSR frequency. The assembly process resulted in 25965 sequences for P. infestans. Annotated transcripts were not subjected to further treatment, and along with the consen- sus ESTs were used for the SSR survey. The frequency of repeat motifs in the consensus EST sequences and annotated transcripts was assessed. A first analysis was performed with only the consensus ESTs from P. infestans; more than 50% of the SSRs identified were (A)n , which is an over-representation with respect to the other mononucleotide repeats as well as to other motifs. Although mononucleotide repeats are common in genomic DNA and can be valid SSRs, most of those present in expressed sequences are the result of nucleotide additions by RNA polymerase and are not present in the genomic DNA template (e.g. poly-A tails; [34]). Therefore, the analysis of monomers was excluded and only motifs with repeats of 2 to 6 bp (for ESTs and transcripts) were included in this study. Both perfect and compound SSRs were selected with a minimum acceptable length of 12 bp for di, tri and tetra-nucleotide motifs. Only SSRs with a minimum of three repeats were included in the analyses of penta- and hexa-nucleotide repeats. The total counts, frequencies and comparisons of SSRs (2–6 bp) in each set of sequences are summarized in Tables 1, 2, 3. P. infestans had the most sequences analyzed but showed the lowest total SSR count and percentage of SSR-contain- ing sequences (Table 1). However, this result has to be considered wih caution since the total size (Mb) of the sequences examined for P. infestans is approximaely half that of the other organisms. To compare the organisms more realistically, another approach was required: namely, taking the total length of each set of sequences analyzed as a reference. Thus, total relative abundance and total relative density were calculated (Table 1). Statis- tically significant differences (P < 0.05) were found between P. infestans and P. sojae and between P. sojae and P. ramorum when we compared the total sequence lengths (bp) contributed by SSRs with respect to the total megabases of examined sequences (relative density), but there was no difference between P. infestans and P. ramo- rum. The same differences were found when perfect and compound SSR proportions were compared (data not shown), demonstrating that SSR content is not in agree- ment with the phylogenetic distances between these organisms [6]. The numbers of SSR-containing sequences and sequences containing more than one SSR were com- pared only between P. sojae and P. ramorum because of the different types of source sequences (ESTs for P. infestans and transcripts for P. sojae and P. ramorum). All comparisons showed statistically significant differences (data not shown). These results might imply that the net SSR content in transcript regions of Phytophthora species could be variable, and again, not directly related to phyl- ogenetic distance. The total numbers of all types of microsatellite motifs are shown in Table 2 and Figures 1 and 2. All three sequence sets contained SSRs that were mainly trinucleotide repeats (> 60%), while the dinucleotide repeats represented less than 8%. This agrees with results from other eukaryotes, where trinucleotide repeats are overrepresented in coding sequences in comparison with dinucleotide repeats [35,36]. Hexanucleotide repeats constituted the second most frequent motif in P. sojae and P. ramorum, differing markedly from P. infestans, where this motif has one of the lowest percentages. In general, statistically significant dif- ferences were not found between P. sojae and P. ramorum (proportions test, P > 0.05), showing that in contrast to the net SSR content, SSR distributions could be related to phylogenetic distances among Phytophthora species. Rela- tive abundance and relative density allowed the similari- ties and differences among SSR distributions to be represented graphically (Figures 1 and 2). The high rela- tive abundance and density values observed for tri- and hexa-nucleotides might correlate with coding region sta- bility ([37]; see discussion). The fifteen most frequent motifs were analyzed for all three organisms in terms of percentage and total counts (Table 3). Most of the motif types were present, and the five most frequent motifs were the same, in all three Phy- tophthora species; however, more motifs were shared between P. sojae and P. ramorum (~80%) than between these two species and P. infestans (~46%). The most com- mon trinucleotide repeats in all cases were (AGC/CGT), (ACG/CTG) and (AAG/CTT); the most common dinucle- otide repeat for P. sojae and P. ramorum was (CG/GC), in complete contrast to P. infestans, where this motif was least frequent. Tetra-, penta- and hexa-nucleotide repeat motifs showed no clear trend among the three organisms. In general, these results and those from the SSR distribu- tions, reflect the more close relation between P. sojae and P. ramorum at the sequence level. Page 3 of 11 (page number not for citation purposes)