Genet et al. BMC Genomics 2011, 12:314 http://www.biomedcentral.com/1471-2164/12/314 Page 4 of 8 12000 10000 re 8000 b uNm6000 S EB 4000 2000 0 10038 451 136 91 33 1 - 10 11 - 50 51 - 100 101 - 150 151 - 200 BLAST hit number Figure 1 Low complexity sequences identified through BLAST search of masked BES against themselves. × axis represents the distribution of BLAST hits. Y axis represents the number of BES. Development of microsatellite markers A total of 6,848 microsatellites were identified in 6,196 BES reads (Table 2). Approximately 56% of the microsa- tellites (3,854) were suitable for PCR primers design as they were flanked by sequences of at least 50 bp. We were able to define 2,061 primer-pairs (~30%) from 1,923 distinct BES as more than one microsatellite can be detected in a single BES. Di-nucleotides were the most abundant repeat motif (65.1%) followed by tetra-nucleotides (10.3%) (Table 2). The most abundant di-nucleotide repeats were TG/CA accounting for 19.5%. AC/GT and GA/TC repeats Table 2 Distribution of Simple Sequence Repeats in trout BAC end sequences. Number Repeat Type Number accounted for 17.9% and 8.8%, respectively. Polymorph- ism and usefulness of the BES microsatellites for linkage analysis and genetic mapping was assessed in the USDA-NCCCWA panel of five families [9] using 193 markers (Additional file 2). Out of the 193 loci tested, 126 (65.3%) were polymorphic with allele numbers ran- ging from 2 to 12; 57 (29.5%) failed to produce a speci- fic PCR product and 10 (5.2%) were monomorphics. Fifteen microsatellite loci were duplicated, of which nine were useful for linkage mapping. These results show that most of the microsatellite markers developed from BES are polymorphic in the NCCCWA reference families and thus can be used for direct integration between the genetic and physical maps of the rainbow trout genome [38]. Homology with other fish genomes We investigated the BES sequence homology and gene content by computational and manual annotations. Of the 176,485 high quality BES reads, 97,846 BES reads (55%) had more than 100 base pairs of contiguous non repetitive sequences and were used for assessing genome syntenies by BLASTN and BLASTX similarity searches against the ENSEMBL genome and peptide databases of zebrafish, medaka and stickleback. The fractions of the 97,846 non-repetitive trout BES reads that had signifi- cant BLASTN hits against the zebrafish, medaka and stickleback genome databases were 15%, 16.2% and 17.9%, respectively, while the fractions of the non-repeti- tive BES reads that had significant BLASTX hits against the zebrafish, medaka, and stickleback protein databases were 10.7%, 9.5% and 9.5%, respectively (Tables 3 and 4). Compared to similar analyses that used BES from non-model fish species to construct “In-silico“ compara- tive genome maps [39-41], the sequence homologies between rainbow trout and the model species were low, Table 3 Rainbow trout BAC end sequences BlastN statistics Medaka Stickleback Zebrafish Total BES with hits BES with unique hitsa BACs BAC with one end Paired BAC-ends Paired BAC-ends with unique hits Unique hits paired BAC-ends matched on the same chromosome Also identified by BlastX Microsynteny identified 15,896 15,379 14,206 845 792 418 85 307 17,499 15,612 15,467 1,016 812 566 105 421 14,716 13,868 13,304 706 632 360 105 176 a The bioinformatic filtering criteria used for identifying ‘unique’ hits are described in the methods section. Repeat Type Monomer A/T C/G Dimer TG/CA AC/GT GA/TC TA/TA AG/CT AT/AT Trimer AAC/GTT CAT/ATG AAT/ATT ATA/TAT TTA/TAA Other Tetramer ACAC/ GTGT TGTA/ TACA ATAC/ GTAT CTCA/ TGAG 71 1 1346 1238 607 505 498 311 5 13 36 36 24 55 26 66 23 25 Tetramer CACA/TGTG TCTG/CAGA TATG/CATA AGAC/GTCT GAGA/TCTC ACAT/ATGT AGAA/TTCT Other Pentamer TCAAA/ TTTGA ATTTG/ CAAAT Other Hexamer Heptamer Octomer Nanomer Decamer Total 38 37 26 38 24 22 17 370 19 15 92 356 80 278 144 478 6,920