Expressed Sequenced Tags (ESTs) with microsatellites, primers and their sequences
White spruce EST sequences were downloaded from the NCBI dbEST. The sequences were searched for the presence of microsatellite repeat motifs using Repeatmasker (http://www.repeatmasker.org). Attempts were made to select sequences whose gene function was annotated. Forty-seven sequences containing dinucleotide, trinucleotide, tetranucleotide and complex repeats were selected (Table 1; Table S1) for primer design. The number of sequences containing dinucleotide, trinucleotide, tetranucleotide, and complex repeats was 22, 14, 3, and 8, respectively (Table 1; Table S1). The TA repeat was the most abundant among the dinucleotide microsatellites. One sequence (RPGSE45) contained microsatellite repeats at two different places and two primer pairs were designed from this EST sequence. The primers for 48 microsatellite sequences were designed using Primer 3 program [16]. However, only one of the two primer pairs developed from the RPGSE45 sequence was used. The information on the source of the EST sequences, GenBank accessions, primer sequences and gene annotations are provided in Table 1 and Table S1. For each microsatellite marker, the M13 universal primer sequence labeled with either 700 or 800 IRDyeTM (Li-Cor, Lincoln, Nebraska, USA), was added to the forward primer for detection of amplicons on Li-Cor 4200 automated sequencer (Li-Cor, Lincoln, Nebraska, USA).
PCR amplification, primer optimization and microsatellites visualization
PCR amplification, microsatellite primer testing, and primer optimization were carried out using four black spruce individuals, including two parents of a genetic mapping population. DNA was extracted from these samples using a Qiagen DNeasy Plant mini kit (Qiagen Canada, Mississauga, Ontario, Canada) and then prepared for downstream analysis.
Polymerase chain reactions (PCRs) were performed in a 10 μl mixture containing 10-20 ng of purified spruce DNA, 2 μg BSA, 1x Taq buffer, 0.2 mM of each dNTP, 0.025U of Taq DNA polymerase (MBI Fermentas, Waltham, Massachusetts, USA), 0.1 μM of reverse primer, 0.1 nM of M13 forward primer labeled with either 700 or 800 IRDyeTM (Li-Cor, Lincoln, Nebraska, USA), and 1.5 mM MgCl2. All PCRs were carried out using a PTC-200, Peltier Thermal Cycler (MJ Research, Waltham, Massachusetts, USA). Each PCR cycle had the following conditions: initial denaturation at 94°C for 5 min followed by 39 cycles each of 94°C for 30 sec, varying annealing temperatures ranging from 50°C to 65°C (determined using a 12-column gradient) for 30 sec, 72°C for 1 min, and a final extension at 72°C for 6 min.
Amplification products were denatured and electrophoresed through a 6.5% polyacrylamide gel run on a Li-Cor sequencing system IR 4200 (Li-Cor, Lincoln, NE, USA) with IRDye 700 and 800 labelled size standards (IR 50-350bp). Each gel was run for 2.5 h with the run parameters of 1400V, 40mA, 40W, 50°C plate temperature, and 16-pixel depths for collection of TIFF image files. The genotypes of individuals were scored manually, and the size of alleles was determined by using SAGA Generation 2 software (Li-Cor, Lincoln, Nebraska, USA). Alleles at a microsatellite locus were identified and named by their molecular size.
Informativeness, characterization and inheritance of microsatellite loci
We evaluated the informativeness of 25 microsatellite loci which could be amplified and resolved by genotyping 20 individuals each of white spruce and black spruce by calculating the number of alleles and their frequency, observed heterozygosity (HO), and expected heterozygosity (HE) using the PopGene software [17]. We also calculated polymorphism information content (PIC) for each locus as ![](data:image/png;base64,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)
where Pi is the frequency of the ith allele in 20 individuals of a species examined. Thus, PIC and HE calculations and estimates are the same. Thirteen white spruce samples originated from seven populations located at five sites in Saskatchewan: Scales Lake, Timber Cove, Snowfield Road, Prairie River, and Tee Pee Creek [details are provided in 18,19]. Seven white spruce samples originated from seven sub-populations (compartments) of the EMEND (Ecosystem Management Emulating Natural Disturbance) project located in northern Alberta approximately 90 km northwest of Peace River [20]. The black spruce samples originated from two populations at Pine Falls, four populations at Bissett, one population at The Pas and four population at Snow Lake sites in Manitoba, Canada [described in 21]. Inheritance of the polymorphic microsatellite markers was examined in a controlled cross from a three-generation outbred pedigree of black spruce [described in 22], consisting of the parents and their 90 progenies. χ2 goodness-of-fit tests were performed to test the inheritance and segregation patterns of the microsatellite DNA markers.