BMC Genomics 2006, 7:245 http://www.biomedcentral.com/1471-2164/7/245 80 70 60 )50 % ( y cn40 e u q re30 F 20 10 0 Table 4: The longest SSR motifs found in EST consensus and transcript sequences for the three oomycetes analyzed. 3 4 5 6 1 11 12 >12 7 8 9 No. of repeats (FFbrilegaqucukre)e,nP3c.ysoojfaree(pgeraety)unanitdnPu.mrabmerosruomf S(SwRhsitfer)om P. infestans Frequency of repeat unit numbers of SSRs from P. infestans (black),P. sojae (grey) and P. ramorum (white). P. ramorum, respectively, and their SSR distributions were characterized. They included enzymes such as cell wall degrading enzymes (cutinases, glucanses, polygalacturo- nases, pectate lyases and cellulases), elicitins and aviru- lence homolog proteins with conserved RXLR motifs, and other secreted proteins potentially related to pathogenic- ity [7,40]. SSRs were also surveyed in two additional sequence sets corresponding to constitutively expressed genes: ribosomal and housekeeping genes such as actin, cytochrome P450-like protein and NADH hydrogenase. The results showed that the percentage of SSR-containing sequences in the pathogenicity factors was not signifi- cantly different from that in the ribosomal and house- keeping genes (Table 5). The scarcity of SSRs in the P. infestans P. sojae P. ramorum di TA (28) TG (28) AC (24) AT (24) AG (28) AG (28) CT (18) CT (18) AG (14) CG (14) AG (12) AC (12) a Canonical notation was used for all SSRs P. infestans P. sojae P. ramorum tri ATT (69) ATT (33) AAG (30) AAC (27) AAG (66) AGG (57) AAG (51) AGG (48) AGC (45) CGT (30) AAG (30) AGG (30) putative pathogenicity factors suggested that SSR length variation has no significant influence on the mutation rate in these sequences. Primer design for EST-SSRs and databases Sequences flanking microsatellites from each of the three organisms were used to develop primer pairs using Primer3 software [41]. For this first survey, the parameters were not stringent since a high number of sequences were analyzed. We produced primer pairs for 61.44%, 87.47% and 88.97% of the SSRs from P. infestans, P. sojae and P. ramorum respectively; not all the SSRs were located in positions suitable for optimum design. Three different primer pairs were generated for each SSR and deposited in the developed database [30] with their sequences, the consensus EST or transcript sequence (with the original sequence ID) and a brief description of the amplifiable SSR. These primers can be used to amplify the correspond- ing SSR region for diverse applications, so they constitute a publicly available resource for future research. Of the three primer pairs, only the first (the best score) was used to analyze transferability. in silico analysis of transferability Primer pairs for each SSR locus were assayed in silico for cross-transferability. Primers for each organism were aligned against the consensus ESTs or transcript databases from the other two organisms. Three criteria were estab- lished to filter the comparisons: (i) high degree of similar- ity (> 90%) between primer and aligned sequence; (ii) primers aligning with only one sequence containing an SSR; (iii) a hypothetical PCR product size longer than 100 bp. Of all the primers designed (8739), 7.5% appeared transferable between at least two species. Not surprisingly, most of the virtually transferable primers were found Repeat Type a tetra CAAG (20) AAAT (20) ATTA (20) ATAA (16) AAGA (32) GCCT (24) GCCT (24) AGCC (24) CTGG (16) CTTC (16) CAGC (16) GTGC (16) penta ATTTT (20) AAAGA (20) CGTGG (15) TTGTT (15) CAAGC (30) GTGTA (25) TCTCA (20) AGAGC (20) CAAAA (15) AGCGC (15) CAAGC (15) CCAAG (15) Hexa CCTGCA (36) GTTGAG (30) CAGCAA (24) AGCAGG (24) TCGGCA (144) TCTACT (96) GCTACG (54) AAGACC (54) CAGGAG (90) AGCGAC (90) GAAGAC (48) CGAGTC (42) Page 6 of 11 (page number not for citation purposes)