Lukacs et al. BMC Genomics 2010, 11:154 http://www.biomedcentral.com/1471-2164/11/154 The amplification products were analyzed using an ABI 377 DNA sequencer. The inheritance patterns of two of these microsatellite markers, Ssa10083BSFU from 114I13 and Ssa10084BSFU from 184H23, were examined in the SALMAP mapping families. Pair-wise linkage analysis was performed using the LINKMFEX software package with a LOD score 4 threshold [64]. Table 3 show the pri- mer sequences of genetic markers used to place 114I13 and 184H23 on the Atlantic salmon linkage map [17]. Gene expression analysis The study was conducted in agreement with the provi- sion enforced by the National Animal Research Author- ity (NARA) [65]. mRNA was extracted from Atlantic salmon tissues of 3 fishes (foregut, hindgut, head kidney, gills, spleen, liver, eye, tongue, skin, muscle, heart, brain and blood) using QuickPrep micro mRNA Purification Kit (GE Healthcare Life Science). 1 μl of mRNA sample was used for quantification with Nanodrop spectrometer (Nanodrop Technologies, DE). All samples were DNase treated using Turbo DNA-free™ (Ambion, Austin, TX, USA). Gene specific PCR primers (Table 3) were designed manually for the amplification of approxi- mately 100-150 bp fragments and synthesized by ProO- ligo (Paris, France). The amplicons were, when possible, placed over introns and product size and specificity was confirmed by agarose gel electrophoresis (Gel logic 200 Imaging system, Kodak) and sequencing. cDNA synth- esis were performed with Ready-To-Go T-Primed First Strand Kit (Amersham, USA) Quantitative real-time PCR was conducted on an ABI7700HT (Applied Biosys- tems, USA). Reactions were performed in 20 μl includ- ing 1 μl cDNA (~6 ng of mRNA) using PowerSYBR Green PCR Master Mix according to the manufacturer’s instructions (Applied Biosystems). PCR parameters were 95°C for 10 min, followed by 40 cycles consisting of 95°C for 15 s, 60°C at 30 s and 72°C at 60 s. A dissocia- tion analysis was performed for each sample to check for unspecific amplification. Relative expression of mRNA in relation to the housekeeping gene elongation factor 1a (EF1A) was calculated using the ΔCT method [66]. Data from real-time RT-PCR are presented as the Mean Relative Expression by calculation of mean 2-ΔCT × 100 for the three fishes used, with three independent samples from each tissue from each fish. Additional file 1: Linkage groups of SAA and UHA. The positions of SAA (Ssa10083BSFU) and UHA (Ssa10084BSFU) in bold and underlined on linkage group 10 and linkage group 14, respectively of the merged Atlantic salmon female map [17]. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 154-S1.JPEG ] Page 15 of 17 Additional file 2: Phylogeny of teleost MHC class I sequences. Phylogenetic tree analysis by NJ method for full-length amino acid sequences. Consensus trees were based on 1000 bootstrap replications and reported with the bootstrap support values (in percent) indicated above the respective nodes. Sequence references are as follows:Auha-UA [Genbank: AAD37813], Dare-UBA*01 [Genbank: CAA86732], Dare-UAA*01 [Genbank: CAA86731], Dare-UDA*01 [Genbank: AAF20178], Dare-UEA [Genbank: AAH53140], Dare-ZE*0201 [Genbank: CAD12790], Dare-L [Genbank: CAD56801], Furu-UBA [Genbank: AAC41236], Gaac-UAA*01 [Genbank: ABN14358], Gaac-UBA*01, Genbank: ABN14357], Icpu-SAA [Genbank: CK423282], Icpu-UAA [Genbank: AAD08650], Icpu-UBA [Genbank: AAD08648], Icpu-UCA [Genbank: AAD08647], Onmy-SAA [Genbank: AF091779], Onmy-UBA [Genbank: AF287483], Onmy-UCA [Genbank: BAD89552], Onmy-UDA [Genbank: AY523666], Onmy-UEA [Genbank: BAD89553], Onmy-UGA [Genbank: AAP04358 ], Onmy-LAA [Genbank: ABI21842 ], Onmy-LBA [Genbank: ABI21844], Orla-UAA*0101 [Genbank: BAD93265], Orla-UBA*0201 [Genbank: BAB83850], Orla- UCA*0101 [Genbank: BAB63957], Orla-UDA*0201 [Genbank: BAB83843], Orla-UEA*0201 [Genbank: BAB83837], Paol-UA1 [Genbank: BAD13367], Paol-ZE [Genbank: BAD13366], Pore-UA [Genbank: CAA90791], Sasa-LBA [Genbank: DY733800 and GO062643], Sasa-UBA*0301 [Genbank: AAN75116], Sasa-UBA*1001 [Genbank: AAN75118 ], [Genbank: ABQ59666], Sasa-ZAA*0101 [Genbank: DQ099914], Teni-UA [Genbank: CR724171], Teni-ZE [Genbank: CAF90807], HLA-A2 [Genbank: AAA76608]. Sasa-UDA, Sasa-UGA, Sasa-UHA1, Sasa-UHA2, Sasa-ULA, Sasa-SAA, Sasa-ZAA*0201, Sasa-ZBA, Sasa-ZCA are described in this paper. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 154-S2.PPT ] Acknowledgements Evelyn Davidson and Wiliam S. Davidson (Department of Molecular Biology & Biochemistry, Simon Fraser University, Burnaby BC, Canada) for help with mapping of BAC clones to linkage groups. The present study was supported by The National Programme for Research in Functional Genomics in Norway (FUGE), The Research Council of Norway, and by NSERC, Genome Canada and Genome BC. Author details 1Department of Basic Science and Aquatic Medicine, Norwegian School of Veterinary Science, Oslo, Norway. 2Centre for Ecology and Evolutionary Synthesis, Dept of Biology, University of Oslo, Norway. 3Department of Biology, University of Victoria, Victoria BC V8W 2Y2, Canada. 4Department of Molecular Biology & Biochemistry, Simon Fraser University, Burnaby BC, Canada. Authors’ contributions MFL and HH: Performed library screening and BAC restriction mapping, sequencing, sequence data analysis, annotations, real time expression and drafted the manuscript; HGB, MBS and LM: Performed library screening and sequencing; KPL: Design of the primers and mapping of BAC clones to linkage groups; BFK: Contributed to the planning and directions; UG: Contributed to planning, design, direction and analysis; All authors read and approved the final manuscript. Received: 11 September 2009 Accepted: 5 March 2010 Published: 5 March 2010 References 1. Ohno S:Evolution by gene duplication New York: Springer-Verlag 1970. 2. Rodgers JR, Cook RG: MHC class Ib molecules bridge innate and acquired immunity.Nat Rev Immunol 2005, 5:459-471. 3. Dijkstra JM, Katagiri T, Hosomichi K, Yanagiya K, Inoko H, Ototake M, et al: A third broad lineage of major histocompatibility complex (MHC) class I in teleost fish; MHC class II linkage and processed genes.Immunogenetics 2007, 59:305-321.