A total of 167 tissue samples were collected from wild boars hunted in Portugal (36 males and 5 pregnant females bearing 31 offspring), Spain (17 males and 5 pregnant females bearing 24 offspring) and Hungary (15 males and 5 pregnant females bearing 29 offspring). All samples used in this work came from dead hunted animals. The dead of the animals did not result for this work, but from legal game hunting activities. Samples were collected, at the end of the day, from dead hunted animals after veterinary inspection. The tissue samples were extracted with JETQUICK Tissue DNA Spin Kit (Genomed, GmbH) according to the manufacturer’s protocol. A total of 27 fluorescent-labeled microsatellite markers were initially tested with a small panel of wild boar samples (n = 16), after a thorough selection based on available bibliographic data (ref. [
14-
19]; Table
). Some of the analyzed markers were discarded either due to low levels of polymorphism (less than 3 alleles), lack of multiplex assay robustness, insufficient information content and difficulty to amplify or score. Finally, a total of 14 loci were chosen and optimized in two multiplex panels each containing 7 microsatellites (Table
). PCR amplification were carried in two independent reactions with the same procedure – a total volume of 10
μl containing 10
ng of genomic DNA, 10
mM of primer mix (Table
), Qiagen Multiplex PCR Master Mix (QIAGEN GmbH, Hilden) and water. The reaction conditions were as follow: (1) an initial denaturation at 95°C for 15
minutes; 2) 10
cycles of 95°C for 30
seconds, 60-56°C (ΔT −0.5°C) for 90
seconds and 72°C for 45
seconds; (3) 22
cycles of 95°C for 30
seconds, 56°C for 90
seconds and 72°C for 45
seconds; (4) 8
cycles of 95°C for 30
seconds, 53°C for 90
seconds and 72°C for 45
seconds, and (5) a final extension step on 72°C for 30
minutes. The samples were then tested in 2% agarose gel and their concentration normalized.
| Table 2Characterization of the STR primer sequence, fluorescent dye used, size range, chromosome location and reference |
The multiplex products were added to a mixture of Hi-Di™ formamide and size standard (Gene Scan™ 500 LIZ size standard) and run in a 3130 XL Genetic Analyzer (Life Technologies) sequencer. GENE MAPPER v4.0 (Applied Biosystems, USA) software was used to analyze the resulting electropherograms in order to identify the obtained alleles. Data quality assessments were scattered along the process either in the form of negative controls (e.g., to exclude contamination problems), or in a final step consisting of a re-amplification and genotyping of randomly chosen samples (10%) to insure a perfect match in the obtained results [
20].
All the data analyses were performed independently for each population and with the exception of maternity and paternity assignment and null alleles, were accomplished using only the adult individuals. The software GENALEX v. 6.41 [
21] was used to calculate the deviations from Hardy-Weinberg Equilibrium proportions (HWE), the probability of identity and the power of exclusion for the loci combinations when both parents are known, when one of the parents is known and for two putative parents. Cervus v. 3.0 [
22] software was used to analyze the number of alleles, observed and expected heterozygosity, combined non-exclusion probabilities (for the first parent and second parent, parent pair, identity and siblings identity) and the estimated null allele frequency (including mothers and offspring). Genepop v.1.2 [
23] software was utilized to calculate the gametic disequilibrium (1000 dememorization steps, 10 batches, 1000 interactions per batch) between alleles of different loci and for each population individually. A maximum-likelihood method implemented in COLONY v. 2.0 [
12,
13] was then used to calculate the assignment probabilities in parentage and sib-ship analyses.
1
