Genomic DNA was extracted from frozen leaf material of apricot (Prunus armeniaca) and cherry (P. persica) using a modified CTAB method , and from freeze-dried leaf material of barley (Hordeum vulgare) and bread wheat (Triticum aestivum) as described by . The apricot lines used were Hasanbey, Trevatt, Rivergem, Patterson, 16822 and 6431. The cherry lines were Sir Douglas, Dame Roma, Sir Tom, Sir Hans, Dame Nancy and Sir Don. The barley lines were Alexis, Chebec, Clipper, Flagship, Harrington, Haruna Nijo, Sahara3771 and Sloop. The bread wheat lines were Barunga, VPM Cook, Chinese Spring, Gabo, Norin10, Olympic, Opata85 and WI7984 (a synthetic hexaploid wheat).
Published primer sets for SSRs and sequence-tagged-sites (STSs) harbouring SNPs were synthesised with generic non-complementary nucleotide sequences at their 5'-ends. Specifically, the forward and reverse primer for each marker was synthesised with the nucleotide sequence 5' ACGACGTTGTAAAA 3' and 5' CATTAAGTTCCCATTA 3', respectively. Primer aliquots for each marker were prepared by mixing equimolar amounts of appropriate forward and reverse primers in 1× TE (1 mM EDTA, 10 mM Tris-HCl, pH 8.0), and are hereafter referred to as locus-specific primers. Two generic tag primers, tagF and tagR, with the sequences 5' ACGACGTTGTAAAA 3' and 5' CATTAAGTTCCCATTA 3', respectively, were also synthesised. The tagF primer was labelled at its 5'-end with one of the following fluorescent dyes: VIC, FAM, NED and PET (Applied Biosystems).
The optimal concentration of locus-specific primer required to amplify the target sequence was determined empirically. Initially 20, 30, 40 and 80 nM of locus-specific primer was tested. PCR products were separated on a GelScan2000 instrument (see SSR analysis section) or on 2% agarose gels stained with ethidium bromide. The optimal primer concentration was determined by visual inspection as the strong amplification of a PCR fragment of the expected size. In instances where it was desirable to improve PCR specificity and yield, additional locus-specific primer concentrations were tested.
Multiplex-ready PCR assays
The amplification of published SSRs and sequences harbouring SNPs by uniplex and multiplex PCR was performed under identical reaction conditions. PCR was performed in a 6 μl reaction mixture containing 0.2 mM dNTP, 1× ImmoBuffer (Bioline) (16 mM (NH4)2SO2, 0.01% Tween-20, 100 mM Tris-HCl, pH 8.3), 1.5 mM MgCl2, 100 ng/μl bovine serum albumin Fraction V (Sigma Aldrich), 75 nmol each of dye-labelled tagF and unlabeled tagR primer, 50 ng genomic DNA, 0.15 U Immolase DNA polymerase (Bioline), and an appropriate concentration of locus-specific primer (see Additional File 1). For multiplex PCR, locus-specific primers for several markers were added to each reaction at the optimal concentration determined in uniplex assays. Due to limited marker choice, some redundant use of primer sets amplifying SSRs and sequences harbouring SNPs was required to construct the multiplex panels. Following an initial denaturation step of 10 min at 95°C to heat activate the DNA polymerase, PCR was performed for a total of 65 cycles with the profile: 30 s at 92°C, 90 s at 50°C, 60 s at 72°C for five cycles. The next 20 cycles were 30 s at 92°C, 90 s at 63°C, and 60 s at 72°C, followed by 40 cycles with 15 s at 92°C, 30 s at 54°C, and 60 s at 72°C, and a final extension step of 10 min at 72°C. The purpose of the initial five cycles with 50°C annealing was to improve the amplification efficiency of published primer sets with low (< 50°C) annealing temperature. Preliminary studies showed that the amplification yield of such primers could be improved without significantly increasing the locus-specific primer concentration. The initial five cycles of 50°C annealing can be eliminated if only primer sets with ≥ 50°C annealing temperature are used.
Conventional PCR assays
The amplification of published SSRs and sequences harbouring SNPs by uniplex and multiplex PCR was performed under identical reaction conditions. PCR was performed in a 6 μl reaction mixture containing 0.2 mM dNTP, 1× Tfi PCR buffer (Invitrogen) (25 mM KCl, 75 mM (NH4)2SO4, 1 mM DTT, proprietary stabilizers, 50 mM Tris-HCl, pH 8.4), 1.5 mM MgCl2, 50 ng genomic DNA, 0.15 U Platinum Tfi DNA polymerase (Invitrogen), and 200 nmol each of standard length forward and reverse primer (see Additional File 1). The forward primers of published SSRs were labelled at their 5'-end with the fluorescent dye HEX (Applied Biosystems). For multiplex PCR, each primer set was added at a concentration of 200 nmol. Following an initial denaturation step of 2 min at 94°C to heat activate the DNA polymerase, PCR was performed for a total of 50 cycles with the touchdown profile: 30 s at 92°C, 60 s at (Ta+10)°C, 60 s at 72°C, where Ta was 50, 55 or 60°C, depending on the optimal conditions reported for the primer set. Following the first cycle, the annealing temperature was reduced by 0.5°C for the next 20 cycles. The PCR was finished with a final extension step of 10 min at 72°C.
Electrophoresis and visualization of SSRs was performed on a GelScan2000 (Corbett Research) and ABI3730 DNA analyser (Applied Biosystems). For analysis on the GelScan2000, PCR products were mixed with an equal volume of gel loading buffer (98% formamide, 10 mM EDTA and 0.5% basic fuchsin as tracking dye), heated for 3 min at 95°C, chilled quickly on ice and separated on a 4% sequencing gel . For ABI3730 analysis, a standardized multi-pooling procedure was used to prepare SSR products amplified by multiplex PCR for electrophoresis. PCR products were diluted with five volumes of sterile water, pooled together at a ratio of 2:2:1:2 for VIC:FAM:NED:PET to give a final 20 μl volume, and desalted by ultra-filtration using an AcroPrep 384 filter plate with 10 kD Omega membrane according to the manufacturer's instructions (PALL Life Sciences). The desalted SSR products were resuspended in 25 μl of sterile water. Three μl of the resuspended SSR product was added to 8 μl of deionised formamide containing 0.8 μl of GelScan500 LIZ size standard (Applied Biosystems). The mixture was heated uncovered at 80°C for 5 minutes to evaporate the water and electrophoresed according to the manufacturer's instructions. This multi-pooling procedure resulted in 0.06 μl of each PCR product being electrophoresed. SSR allele sizing was performed using GeneMapper v3.7 software (Applied Biosystems). The pooling of PCR products with different dye-labels at the 2:2:1:2 ratio was to account for differences in the relative fluorescence of each fluorophore.
SNP assays were performed using allele-specific primer extension chemistry and xMAP™ technology (Luminex Corporation) on the BioPlex microsphere-based suspension array platform (BioRad). The assay was performed essentially as described by  and involved: (a) an initial PCR amplification of the target sequences harbouring the SNPs from genomic template, (b) purification of the PCR products to remove residual primer and dNTP, (c) assay of the SNPs using allele-specific primers with oligonucleotide capture probes at their 5'-ends, and (d) detection of the allele-specific primer extension products on the BioPlex instrument.
The multiplexed PCR products were diluted to 50 μl with sterile water and purified by ultra-filtration to remove excess PCR primer and dNTP using a Montage 384 PCR cleanup plate (Millipore) according to the manufacturer's instructions. The purified PCR products were resuspended in 20 μl of sterile water. Five μl of purified PCR product was added to a PCR well and dried by heating at 80°C for 15 min before 5 μl of allele-specific primer extension mix was added, which contained 1.25 μM each of dATP, dTTP, dGTP and biotin-14-dCTP (Invitrogen), 1.25 mM MgCl2, 1 × Tsp PCR buffer, 0.1 U Platinum Tsp DNA polymerase (Invitrogen), and 25 nmol of each allele-specific primer (see Additional File 1). The thermocycling conditions involved an initial denaturation step of 2 min at 94°C to activate the DNA polymerase, followed by 30 cycles of 30s at 94°C, 60s at 55°C, and 2 min at 74°C.
Following PCR, the allele-specific primer extension products were hybridised to fluorescently colour-coded carboxylated polystyrene LabMAP™ microspheres (Luminex Corporation). Each allele-specific primer contained an oligonucleotide sequence (ZipCode) at its 5'-end, allowing hybridization to a complementary sequence (called a cZipCode) covalently attached to the microsphere surface. The hybridization of the biotinylated allele-specific primer extension products for each SNP to their corresponding microspheres was carried out essentially as described by  in a 30 μl reaction volume containing 1× hybridization buffer (200 mM NaCl, 0.08% Triton X-100, 100 mM Tris-HCl, pH8.0) and 500 microspheres of each type. After initial denaturation at 95°C for 5 min and incubation at 37°C for 30 min, the microspheres were washed once with 100 μl of 1× hybridization buffer, resuspended in 50 μl of 1× hybridization buffer and conjugated with streptavidin by adding 250 μg of streptavidin-conjugated R-phycoerythrin (Invitrogen). The microspheres were incubated at room temperature for 15 min, washed once with 100 μl of 1× hybridization buffer, resuspended in 50 μl of 1× hybridization buffer and assayed on the BioPlex microsphere suspension array platform. The BioPlex instrument first identifies the specific microsphere (and thus the SNP locus) based on the microsphere colour then the presence or absence of the streptavidin-biotin conjugate (indicative of the presence or absence of the specific allele). The fluorescence on the surface of the microspheres resulting from the streptavidin label was converted to a median fluorescence intensity (MFI) value based on a minimum of 32 microspheres for each of the SNP alleles assayed. The allele present at each SNP locus was determined using BioPlex SNP Manager software v1.0 (BioRad).