Genet et al. BMC Genomics 2011, 12:314 http://www.biomedcentral.com/1471-2164/12/314 database containing 735 putative repeat elements. This database is useful for repeat masking in salmonid gen- omes. Approximately 59.5% of the BES database in base pairs was masked by this repeat database, providing for the first time an estimate of how much of the rainbow trout genome is composed of repetitive sequences. We detected 6,848 microsatellites in the BES dataset, of which 3,854 presented high quality flanking sequences with more than 50 bp in length. A subset of those were validated and used to construct the first rainbow trout integrated genetic-physical map [38]. The development of those new microsatellite markers will also serve to increase marker densities on current rainbow trout genetic maps and initiate in silico comparative mapping with species whose genomes have been fully sequenced. Paired BAC-ends were used to establish regions of microsynteny between trout and model fish species (zebrafish, medaka, and stickleback). The microsynteny analyses revealed low to intermediate genome homology between rainbow trout and the other fish species. Our findings suggest that due to chromosomal breakage and rearrangements that have occurred during fish genomes evolution, only closely related species like other salmo- nids will be useful for chromosome-wide and genome- wide comparative analyses with rainbow trout. Methods BAC culture and BAC-end sequencing A 10X HindIII bacterial artificial chromosome (BAC) library from a Swanson YY male doubled haploid homo- zygous line was previously constructed [4]. More than half of this library (99,840 BAC clones) was used for BAC-end sequencing. BAC culture and sequencing reac- tions were conducted, as described previously [43]. Briefly, BAC DNA was extracted using a standard alka- line lysis protocol developed by Genoscope (Evry, France). Sequencing reactions were carried out in the Genoscope facility with T7 or Sp6 universal primers, using ABI kit version 3.1. Generated raw sequence files were subsequently processed using the PHRED software [44], vector and bacterial sequences were removed. Q20 values were achieved by setting the sequence quality PHRED score cut-off value to 20. All processed BES were submitted to the EMBL/EBI database with conse- cutive accession numbers of FQ482162-FQ658498 and are available through the web site of the INRA bioinfor- matics group (http://www.sigenae.org/troutBES). Identification of repetitive DNA elements Complex DNA repetitive elements RepeatModeler software was used for identifying repeat elements boundaries and for classifying the newly recon- structed repetitive sequence from the rainbow trout BES data (http://www.repeatmasker.org/RepeatModeler.html). Page 6 of 8 A specific rainbow trout repeat database was con- structed and was named INRA RT Rep1.0. This data- base was subsequently used as a custom file for masking BES sequences using RepeatMasker (http://www.repeat- masker.org/). The Atlantic salmon repeat database was used (file Salmon Raw Repeat DB V1_6 available at http://web. uvic.ca/grasp) for comparison purposes. Identification of microsatellites and simple sequence repeats Microsatellites and other SSR motifs were identified using Tandem Repeat Finder (TRF) software [45]. We examined ten classes of SSRs by using a maximum per- iod size of 10 with default settings. BES containing microsatellites were subsequently masked using Repeat- Masker with INRA RT rep1.0 custom library file: BES harbouring SSRs with at least 50 bp flanking sequences were then selected and forward and reverse primers were designed using Primer3 software [46]. Microsatel- lites and corresponding flanking sequences were sub- mitted to the GenBank STS database with consecutive accession numbers of GF100674-GF100698; GF107484- GF107651; GF107921-GF109647; GF110457-GF110594 and GF110820-GF110822. Assessment of microsatellites polymorphism The polymorphism of 193 microsatellites markers was assessed by genotyping 10 parents from the National Center for Cool and Cold Water Aquaculture (NCCCWA) reference mapping panel [9]. Primers were optimized for amplification by varying annealing tem- peratures and MgCl2 concentrations. PCR amplifications were conducted in an MJ Research DNA Engine thermal cycler model PTC 200 (MJ Research, Waltham, MA) as previously described [47]. Three microliters of each PCR product were added to 20 μL of water, 1 μL of the diluted sample was added to 12.5 μL of loading mixture made up with 12 μL of HiDi formamide and 0.5 μL of Genscan 400 ROX internal size standard. Samples were denatured at 95 °C for 5 min and kept on ice until load- ing on an ABI 3730 DNA Analyzer (ABI, Foster City, CA). Output files were analyzed using GeneMapper ver- sion 3.7 (ABI, Foster City, CA), Assessment of regions of synteny with other fish genomes Sequence homology searches and results filtration Masked BES reads with more than 100 base pairs of con- tiguous non-repetitive sequences were analysed for sequence homology by BLASTN using ENSEMBL DNA databases for zebrafish (Danio_rerio.Zv9.61.dna_rm.tople- vel.fa), stickleback (Gasterosteus_aculeatus.BROADS1.61. dna_rm.toplevel.fa), and medaka (Oryzias_latipes. MEDAKA1.61.dna_rm.toplevel.fa) and for gene content by BLASTX using the ENSEMBL non redundant protein