Survey and analysis of microsatellites from transcript sequences in Phythoptora species: frequency distribution and potential as markers for the phylum Oomycota.

Sources of ESTs and annotated transcript sequences analyzed

EST sequences from P. infestans (81974) were downloaded from the Phytophthora Functional Genomics Database (http://www.pfgd.org/). The annotated transcript sequences available for P. sojae (19276) and P. ramorum (16066) were downloaded from the Department of Energy's Joint Genome Institute (http://www.jgi.doe.gov/).

EST assemblage

An assembling and clustering process was done for ESTs only from P. infestans in order to generate a non-redundat data set. UniVec build # 3.2 was obtained from the NCBI web site FTP directory http://ftp.ncbi.nih.gov/pub/UniVec and the latest repetitions database from http://www.girinst.org/. EST set was masked using the cross-match utility. PHRAP version 0.990329 was used to cluster the ESTs and to generate consensus sequences. Parameters phrap-miniscore 100 -minmatch 50 were used to generate consensus. Detailed description about the clustering process can be downloaded.

Microsatellites search

Consensus ESTs and annotated transcripts were scanned using a local version of the Microsatellite Identification Tool (MISA) available from the Plant Genome Resources Center (PGRC) http://pgrc.ipk-gatersleben.de/. This program searches for both perfect and compound SSRs with 2 to 6 nucleotides in the basic repeat unit. It records repeat numbers and SSR locations inside the EST sequence and deposits these results in an output file.
SSR redundancy was minimized by counting only a single match when there was more than one record for the same SSR locus. Minimum SSR length was determined by the number of repeats, which were (2/6) (3/4) (4/3) (5/3) (6/3); the first digit refers to the SSR repeat type and the second to the minimum number of repeat units (Table 1). For example, (2/6) means that a motif consisting of 2 bp and should have at least 6 repeats to be considered a microsatellite in our analysis.

Primer design

P3_in.pl and P3_out.pl perl scripts, which complement the MISA program, were adapted and used for automated selection and transfer of SSR-containing sequences from the database to the Primer3 program. Parameters used for the Primer3 program were as follows: optimal Tm of 57ºC with a minimum and maximum of 50ºC and 64ºC respectively, and 50-75% GC content. The probability of dimer or hair-loop formation was low. The size of the PCR product is expected to be between 50 and 300 bp and no secondary structure.

Database IDs meaning

IDs implemented in this database for sequences from P. sojae and P. ramorum are the original IDs used in the Department of Energy's Joint Genome Institute (http://www.jgi.doe.gov/) database. IDs implemented for consensus ESTs from P. infestans were assigned for us since the original sequences had to be assembled. Original IDs from Phytophthora Functional Genomics Database (http://www.pfgd.org/), which conform each consensus sequence are available