Sadd et al. BMC Genomics 2010, 11:110 http://www.biomedcentral.com/1471-2164/11/110 genome may be spurious, it is possible that they are the result of either incomplete gene annotation in the A. mellifera genome or evolution of pseudogenes in the Apis lineage. In an attempt to gain a further insight into these possibilities, from the 20 unique sequences with the longest predicted reading frames we inspected more closely the sequences that had a highly significant “hit” to another insect protein (BLASTX, <1e-20) (Table 3). By “blasting” these proteins against the Apis genome (TBLASTN), we attempted to find reasons that these proteins are not in the set of predicted Apis proteins. All six of the proteins had “best-hits” in the same loca- tion of the genome as the unique sequences had, and visual inspection of alignments revealed premature stop codons were coded for in the Apis sequences in four out of the six cases. Immune genes and pathways While the bumblebee workers used to produce this EST library were not immune challenged, the vast amount of work on immunity and host-parasite interactions in these insects [19,43-45] makes this category of genes nonetheless worthy of further interest. Furthermore, parasites have been implicated in declines of natural and commercial pollinators, such as bumblebees, worldwide [25,46,47], and thus knowledge of genes and pathways involved in immunity and parasite defence is highly important. Based on the annotations of the unique sequences, and in comparison with proposed A. mellifera immune genes [48], we find that 134 unique sequences have “best hits” to proteins from genes characterised as immune related (Additional file 6). These 107 unique sequences in Bombus terrestris match to 67 Apis genes (a similar level of redundancy to that shown with all A. mellifera protein “hits”). In addition, based on the classi- fication by Sackton et al. [49], 101 unique sequences have “best-hits” in D. melanogaster to proteins of 58 immune genes. However, a core set of only 32 unique Page 5 of 9 sequences had both immune classified “hits” in D. mela- nogaster and A. mellifera, with “hits” to 24 and 25 unique genes, respectively. This could be due differential annotation of immune genes in the two organisms, or different divergence from B. terrestris among different genes. The potential immune gene transcripts found span the major described pathways in the invertebrate immune system (Figure 2), and based on these certain compo- nents, we can begin to build a picture of immune defence in bumblebees. We find that there is evidence for the existence and transcription of integral genes in each of the described pathways. Potential EST-derived genetic markers Traditional methods of isolating and characterising genetic markers can be expensive and time-consuming. However, ESTs can be a valuable resource for the initial identification of genetic markers that can subsequently be used in studies of molecular ecology [36]. Simple sequence repeats (SSRs) and single nucleotide poly- morphisms (SNPs) allow population level studies both within the target species and related species. Using MISA [50], a number of SSRs were identified in the unique sequences. We restricted our search for SSRs to di-, tri-, tetra-, penta-, and hexa- nucleotide motifs that were repeated at least six times for di-nucleotide and five times for all other motifs. We found a total of 589 markers found in 507 unique sequences. 397 di- (mean repeat length = 7.65, range = 6-17), 154 tri- (mean repeat length = 5.79, range = 5-17), 26 tetra- (mean repeat length = 6, range = 5-11), 6 penta- (mean repeat length = 5.83, range = 5-7), and 6 hexa- (mean repeat length = 6.33, range = 5-9) nucleotide repeats were found. 61 of the SSRs were found in compound formation with another (maximum of 100 nucleotides separation). While polymorphism remains to be verified for these markers with appropriate laboratory screens, previous levels of polymorphism in EST derived SSRs Table 3 Unique sequences from the 20 longest ORFs that have “hits” to other insect proteins and also “hits” away from protein coding regions in the Apis genome, but are not part of the Apis protein set Project Identification (bom001no-) CL2097Contig1 CL2223Contig1 CL2537Contig1 CL2610Contig1 CL2809Contig1 P0115M15_F Organism “best-hit” Protein Description N. vitripennis N. vitripennis N. vitripennis N. vitripennis N. vitripennis P. humanus corporis XP_001604128.1 NP_001123269.1 XP_001606900.1 XP_001600663.1 XP_001604974.1 XP_002432715.1 similar to F-box protein 28 nucleoporin Ndc1 similar to p53-like protein similar to ENSANGP00000017887 similar to GA18228-PA nuclear pore complex protein nup98 Potential reason for absence from A. mellifera protein set Stop codon Unknown Stop codon Unknown Stop codon Stop codon Unique sequences, from the sequences with the 20 longest ORFs that have “hits” to the Apis genome at least 4’375 bases away from predicted Apis protein coding regions, with highly significant “best-hits” (BLASTX, 1e-20) to an insect protein. These “best-hit” insect proteins were “blasted” against the Apis genome (TBLASTN) and the resulting alignments scanned for potential elements hindering protein production.