BMC Genomics 2006, 7:245 http://www.biomedcentral.com/1471-2164/7/245 Table 5: Distribution of SSRs in three functional categories of genes Sequences examined Number of identified SSRs SSR-containing sequences (%) P. infestans P. sojae P. ramorum Pathogenicity factorsa Ribosomal genes Housekeeping genesb Pathogenicity factorsa Ribosomal genes Housekeeping genesb Pathogenicity factorsa Ribosomal genes Housekeeping genesb 136 44 41 318 52 22 171 50 48 24 2 6 50 11 3 16 3 5 16.91% 5.54% 14% 11.63% 17.30% 13.63% 9.35% 6% 8.33% aPutative sequences of cutinases glucanses, polygalacturonases, pectatoliases, cellulases, elicitins and proteins with conserved RXLR motif were selected for this analysis bPutative gene sequences of actin, cytochrome P450-like protein and NADH hydrogenase were selected for this analysis. between P. sojae and P. ramorum, since these are the two most closely related species. However, 84 (~1 %) primers were found to be virtually transferable among all three species (Table 6). Discussion The present study was designed to create microsatellite databases for P. infestans, P. sojae and P. ramorum, taking advantage of publicly available sequences for these organ- isms. In the case of P. infestans, EST sequences were first assembled and then explored for SSRs. For P. ramorum and P. sojae, annotated transcripts from the available whole genome sequences were used directly to search for SSRs and to analyze the distribution and organization of SSRs in the transcribed regions of these organisms. Approximately 6–22% of the sequences contained SSRs, which shows higher frequencies than previously reported for plant ESTs [25,37,42], fungal endophytes [43] or other higher eukaryotes [8,44]. However, these differences might reflect the different criteria used to select the SSRs. Repeat sequences of at least 12 bp for di-, tri- and tetra- nucleotides, and three or more repeated units for penta- and hexa-nucleotides, were chosen for this study. These lengths were used because they have been considered the Table 6: In silico determination of potential cross-transferability of the SSRs between Phytophthora species Primer transferability a 100% 95–99 % 90–94 % P. infestans – P. sojae P. infestans – P. ramorum P. sojae – P. ramorum P. infestans – P. sojae – P. ramorum 7 6 27 4 21 8 126 19 91 50 282 61 a When an EST or transcript sequence matched primer pairs designed from species 1 and species 2, they were counted as one primer pair. minimum acceptable microsatellite lengths [34] and are efficient thresholds for detecting high levels of polymor- phism [22]. Our results showed that SSR lengths are very restricted in the coding regions of oomycete genomes; approximately 99% of all the SSRs analyzed were shorter than 30 bp. Only a few SSRs had higher numbers of repeat units, perhaps because of their location in well-conserved regions of the genome. Strong evolutionary and func- tional constraints limit the expansion of microsatellite repeats in expressed regions of the genome [45,46], because longer repeats have higher mutation rates and could therefore be less stable [47,48]. Short microsatel- lites are probably generated by random mutations and then expanded by DNA polymerase slippage. Thus, the base composition of a sequence that seeds the evolution of repeats is expected to influence microsatellite density [49,50]. Therefore, the similarities between SSR motifs within oomycetes and between oomycetes and fungi may indicate that specific common sequence composition contributes to the evolution of SSRs. Statistically, there were no quantitative differences in the distributions of SSR motifs between P. sojae and P. ramo- rum. However, marked differences were found when these two species were compared with P. infestans. This is partic- ularly interesting because the frequency distribution of SSRs does not follow a genus-wide pattern; if it is strongly species-dependent, it could indicate evolutionary events specific to the organisms compared. In addition, SSRs derived from exonic regions, which are more conserved than genomic SSRs, might consequently show only minor differences in distribution among related species. Thus, differences in genomic organization could explain the SSR distributions observed in the organisms examined, reflect- ing the phylogenetic distances between them [29,51]. More sequence data on ESTs/transcripts will soon become available for Stramenopiles, as P. infestans, P. capsici, Page 7 of 11 (page number not for citation purposes)