Ethics statement and sample collection
All Siberian roe deer, European roe deer (Leningrad oblast), Eurasian elk and red deer tissue samples were provided by local hunters; animals were not killed for the purpose of this study. Fibroblast tissue cultures of European roe deer (UK), cattle and brown brocket were taken for DNA isolation from the collection of the Comparative genomics group (Cambridge). Table 1 lists the number of specimens and their characteristics.
Cell culture and chromosome preparation
Fibroblast cell lines were established from the ear cartilage tissue of Siberian roe deer individuals with different numbers of B chromosomes as described previously . The cell lines were cultured at 37°C in Dulbecco’s modified Eagle medium (DMEM) and αMEM (Gibco, Invitrogen, Paisley, UK) enriched with 10% fetal bovine serum (Gibco), penicillin (100 units/ml) and streptomycin (100 μg/ml) or kanamycin (100 μg/ml). Before harvest, the cells were treated with 1 μg/ml of ethidium bromide (final concentration) for 2 h and treated with 0.05 mg/ml colchicine (final concentration) for 45 minutes. Preparation of metaphase chromosomes was made according to standard procedures that included a 45-minute hypotonic treatment in 0.56% KCl, fixation in 3:1 methanol/glacial acetic acid, followed by slide preparation and air drying. The number of B chromosomes was analyzed in all C. pygargus samples (at least 50 cells per sample) and we detected no variation in the number of B chromosomes between cells.
B chromosomes of the Siberian Roe deer (CPY_d) have been flow sorted on a FACStar Plus, Becton Dickinson, USA, as described previously [45, 46]. The peak containing B chromosomes was very well resolved and distinct from non-B chromosomal peaks (Additional file 4: Figure S2). We collected B chromosomes into 4 tubes containing 20 μl of water (1,000 chromosomes per tube).
Generation of B chromosome-specific libraries
Chromosome-specific libraries were made by DOP-PCR amplification of flow-sorted chromosomes . DOP-PCR-amplified chromosome-specific DNA was labeled during the secondary PCR by incorporating biotin-16-dUTP (Roche, Switzerland) or Cy3-dUTP (GE Healthcare, UK) . Chromosome specificity of the library was confirmed by FISH (Figure 1).
Selection of B chromosome-specific cDNAs
Total RNA was isolated from several millions of fibroblasts from the sixth passage of the Siberian roe deer primary culture (CPY_d) using the MasterPure RNA purification Kit (Epicentre, UK) according to the manufacturer’s protocol. A double stranded total cDNA library was constructed using the BD SMART™ PCR cDNA Synthesis Kit (BD Biosciences) according to the manufacturer’s protocol. Two rounds of B chromosome-specific cDNA selection were conducted using a method based on previously published selection of hybrids by affinity capture (SHAC) . Briefly, 50 μl of cDNA PCR library (driver) and 1 μl of biotin-labeled B chromosome-specific library mixed with 5 μg of C. pygargus Cot5 DNA (tracer) were denatured for 3 minutes at 95°C. After denaturing the products were adjusted with sodium dodecyl sulfate (SDS) (up to 0.1%) and saline-sodium citrate (SSC) (up to 1 ×) and prehybridized separately at 65°C for 30 minutes to suppress the repetitive elements. Both solutions were mixed together and hybridized overnight at 65°C. The resulting DNA duplexes were captured by streptavidin-coated magnetic beads, Dynabeads® M-280 Streptavidin (Invitrogen, UK) for 20 minutes at 65°C. Magnetic beads were washed twice in 2 × SSC, 0.1% SDS for 5 minutes at 65°C, five times in 0.2 × SSC, 0.1% SDS for 5 minutes at 65°C and once in 0.2 × SSC at 30°C. The cDNAs were released by denaturing the beads in Tris-ethylenediaminetetra-acetic acid (TE) buffer for 5 minutes at 95°C. Selected cDNAs were amplified by PCR with specific primer (provided by BD Bioscience). cDNAs, after the first round of selection were subjected to the second round, to increase the chromosome specificity. The quality of the final CBCLs was controlled by FISH (Figure 2).
Cloning of cDNA fragments
The B-specific cDNA library was cloned using TOPO® TA Cloning® Kit (Invitrogen, UK) according to the manufacturer’s instructions. In all, 50 single colonies were cultured for plasmid DNA isolation; 33 clones with BD primer flanked inserts were selected for sequencing.
Fluorescence in situ hybridization
Probes for fluorescence in situ hybridization were labeled either by direct incorporation of labeled dUTP in PCR (with DOP or BD primers) or using a nick translation kit (Invitrogen, UK) following the manufacturer’s instructions (for bovine BACs). Fluorescence in situ hybridization was performed using a standard protocol [49, 50].
Images were captured and processed using the CytoVision Genus system (Applied Imaging, USA) and a Cohu CCD camera mounted on an Olympus BX-60 microscope, or using Videotest 2.0 Image Analysis System (Saint Petersburg, Russia) and a Baumer Optronics CCD Camera mounted on Axioscope 2 plus microscope (Carl Zeiss, Germany).
Isolation of DNA from bovine BAC clones
DNA was isolated from bovine BAC clones (Additional file 2: Table S1) of the genomic BAC library CHORI-240 using the QIAGEN Large-Construct Kit (QIAGEN, UK).
Isolation of DNA from animal tissues and cell cultures
DNA was isolated from all samples listed in Table 1 using DNeasy Blood & Tissue Kit (QIAGEN) according to the manufacturer’s instructions.
PCR-assisted mapping of the flow-sorted CPY B chromosome-specific library
PCR-assisted mapping was conducted using primers designed from cattle chromosome 3 region (UCSC genome browser on Cow, October 2011 (Baylor Btau_4.6.1/bosTau7)) listed in Additional file 5: Table S2. A total of 20 ng DNA of the Siberian roe deer with B chromosomes (CPY_d), flow sorting-derived CPY B chromosome-specific library and cattle genomic DNA were used as templates. The PCR program included: 94°С for 2 minutes; 30 cycles of 94°С (15 s), Х°С (30 s), 72°С (30 s); and a final extension at 72°С for 5 minutes, where Х corresponds to the respective annealing temperature for each pair of primers. PCR products were analyzed on a 1.5% agarose gel.
The fragment of the PTGFR gene amplified using the NBF and NBR primers (Additional file 6: Table S3) on cattle genomic DNA was used as a reference fragment for the real time PCR calibration. The primer sequences were designed from the genomic regions conserved between cattle and the Siberian roe deer. Real time PCR was performed using standard curve methods  using a C1000 Thermal Cycler (BioRad). C1000 manager software was utilized for the analysis of the results.
The copy number of regions was estimated in the genomes of cattle and Siberian roe deer with different numbers of B chromosomes. Each primer pair was tested by non-template PCR to check the primer-dimer formation in the reactions. A final dissociation step was always performed at the end of each PCR to identify the unique and specific amplification of the target sequence. Additional file 6: Table S3 lists the primers used for real time PCR in the present study. The amplification reactions were run three times. Reactions contained 10 μl of DNA (0.2 μg), 5 μl of primers mix (final primer concentration of 1 mM each), and 10 μl of Power SYBR Green PCR Master Mix (Sintol, Moscow, Russia), in a final volume of 25 μl. Amplifications were carried out in a Thermal Cycler C1000 (BioRad). The reactions started with an initial step at 95°C for 5 minutes. The reaction proceeded with 40 cycles of 95°C for 15 s, 60°C for 20 s and 72°C for 30 s. A final dissociation step was always performed to control the amplicons melting curves.
B-specific cDNA clones were sequenced with standard M13 primers using DTCS Quick Start Master Mix and CEQ 2000 sequencer (Beckman-Coulter) in the Department of Veterinary Medicine (Cambridge, UK). PCR products for sequence analysis of FPGT, TNNI3K and LRRIQ3 genes were generated using primers based on cattle chromosome 3 region (UCSC genome browser on Cow, October 2011 (Baylor Btau_4.6.1/bosTau7)) (listed in Additional file 7: Table S4). For comparative analysis of non-B chromosomal and B chromosome-specific copies we amplified the segments from B chromosome-specific libraries, DNA of individuals lacking Bs and microdissected autosomes. Sequencing was carried out in the Interinstitutional center of DNA sequencing at the Siberian Branch of Russian Academy of Sciences (Novosibirsk, Russia) using an ABI3130×1 Genetic Analyzer (Applied Biosystems Inc., CA, USA) with ABI Big Dye kit according to a standard protocol. Sequence analysis was accomplished using Sequence scanner V1.0 software (Applied Biosystems), Blast and Blat alignment tools [52, 53].
We microdissected several copies of the largest autosome (CPY1) and B chromosomes as described earlier  with some modifications: after incubation in the collection drop with proteinase K and SDS, the sample was transferred to water, denatured and adjusted with all reagents necessary for PCR with T2 primers (Additional file 5: Table S2). In all, 40 cycles were performed (controlled with samples containing other autosomes and total genomic DNA) revealing fragments of interest. The fragments were directly sequenced after ExoSap treatment.
The GenBank  accession numbers for DNA sequences are JN871269 to JN871295.