BMC Genomics 2006, 7:245 http://www.biomedcentral.com/1471-2164/7/245 Pythium ultimum, and Hyaloperonospora parasitica are or will be sequenced in the near future that will provide fur- ther resolution on the evolution of SSR motifs in Oomyc- ete coding regions. The most common SSRs comprised trinucleotide repeat units with low repeat numbers. A wide variety of repeat motifs were represented at high percentages in these trinu- cleotide arrays. The abundance of repeat motifs differed slightly, especially between P. infestans and the other two species; (AGC)n, (ACG)n and (AGG)n were the most abundant triplets in all organisms but their abundances differed among species. This finding was expected, since EST or transcript-derived microsatellites are likely to be conserved in frequency, abundance and distribution across closely-related species [52]. A database search of all possible trinucleotide repeat motifs (>20 bp) showed that (AGG)n, (AAT)n and (ATC)n are relatively common in fungi, but (ACG)n and (CCG)n are relatively rare [53]. Differences in abundance and density among trimeric repeats could be explained by species-specific cellular fac- tors that interact with the motifs and play an important role in generating the repeats [18]. Among plant species, the abundances of different repeat motifs in EST-derived SSRs vary greatly. However, trinucleotide units with low repeat numbers are common features of EST-SSRs [25,37,42,54]. Our results suggest that Phytophthora spp. might have a set of common motifs, as is the case with fungi, whereas the motifs in oomycetes may vary widely, as they do in plants. High dinucleotide repeat abundances in whole genomes have been reported for fungi, Drosophila,Caenorhabditis ele- gans and a subset of plant genomes [36,51,54,55]. Dinu- cleotide repeats have been characterized as being the most important SSRs because of their higher mutation rates [51]. This suggests an explanation for their high abun- dance in genomic regions and low abundance in coding regions, which must be conserved to maintain functional- ity. On the other hand, many studies have reported that trinucleotides are most abundant in coding regions of higher eukaryotic genomes [46,56,57]. Previous studies have shown that trinucleotide repeats predominate in plant EST libraries, supporting our observations on oomycetes [12,58]. Among all SSRs, expansions or dele- tions in coding regions can be tolerated for tri- and hexa- nucleotides, which do not perturb reading frames [36]. This could explain why the longest SSRs in all the organ- isms examined belong to these motif categories, although the three-dimensional structure of a protein translated from a sequence containing such SSRs would not neces- sarily be unchanged. Interestingly, our analyses showed that glutamine is the amino acid encoded by the most highly abundant repeat in P. sojae and P. ramorum: (CAG)n . This agrees with previous reports on Drosophila, C. elegans and yeast [36], in which the same glutamine-coding tri- plet was the most common in tandem triplet repeats. In contrast, glutamic acid was encoded by the most common repeat in P. infestans: (AAG)n . Like glutamine, this is also one of the most common tandemly repeated amino acids in the aforementioned organisms. Katti et al. [59] ana- lyzed all the protein sequences from the SWISS-PROT database for single amino acid repeats, tandem oligo-pep- tide repeats, and periodically conserved amino acids; the results showed that repeats of glutamine, serine, glutamic acid, glycine and alanine seem to be fairly well tolerated in many proteins. Four of these five amino acids were found in our analysis and have also been reported in recent studies of complete genome coding sequences [36]. Such triplet repeat patterns in ORFs of oomycetes and other organisms could reflect functional selection of amino acid reiterations in the encoded proteins. Whole genome analyses have shown that repeat stretches of small/hydrophilic amino acids are more frequent in pro- teins [59]. The expansion of codons encoding such amino acids might be better tolerated than the expansion of hydrophobic amino acid stretches because they probably would not change the three-dimensional protein confor- mation as drastically. Therefore, nucleotide composition might strongly affect the structures and functions of encoded proteins, and it could be a determining force in the selection of SSRs in coding sequences. We also explored the possibility that valuable information for mapping and for diversity or population structure studies could be obtained from these data. For this reason, microsatellite markers were evaluated for in silico cross- transferability among all three species. Many studies on organisms from different kingdoms have led to the devel- opment of markers amplifiable across species [34,44,54], even among Phytophthora species [17]. To our knowledge, this possibility has never been assessed in silico for any organism (including oomycetes). SSRs designed from EST/transcripts sequences are especially valuable owing to their genome location, which implies constraints on length, motif, abundance and flanking regions. The last of these is of particular interest for characterizing interspe- cies genetic diversity using common primers. Therefore, we designed primers for all SSR sets observed by species and identified those that were potentially cross-transfera- ble. This approach was similar to a virtual PCR and pro- vided candidate SSRs for experimental screening. The results revealed a small proportion of primers that could theoretically be used as transferable molecular markers (using a similarity criterion of 100%). This was an unex- pected result because EST/transcript types of sequence are highly conserved and there is a close phylogenetic rela- tionship among the Phytophthora spp. [27]. However, if the similarity criterion for alignment was less stringent (90%), 653 primer pairs were virtually transferable Page 8 of 11 (page number not for citation purposes)