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ŠUMARSKI LIST 5-6/2012 str. 39     <-- 39 -->        PDF

of each primer, 0.2 mm of each dNTP and 1 µl of genomic DNA directly from extraction in a total reaction volume of 10 µl. Amplification conditions, with the exception of the annealing temperature for primer pair M2-30, which was 57 °C, were as described by Heuertz et al. (2001). PCR products were multiplexed together with 0.4 µl of internal size standard GS-400 HD ROX and 12 µl of deionised formamide. Positive and negative controls were used to verify the accuracy of the reactions, amplification conditions and fragment analysis. Fragment analysis was performed in ABIPRISM 310. Sizing and genotyping were carried out using accompanying software GeneMapper.
Data analysis – Analiza podataka
The number of alleles per locus A, number of alleles with frequency equal to or higher than 0.05 A≥ 0.05, number of effective alleles AE, number of private alleles APRIV, proportion of observed heterozygotes HO were calculated using GenAlEx 6 (Peakall and Smouse 2006) and gene diversity HE as well as total gene diversity HT with FSTAT (Goudet 1995). Wright’s inbreeding coefficient FIS, FST (relative differentiation based on allele identity) and RST (relative differentiation based on allele size) and their statistical significance were calculated using SpaGeDi (Hardy and Vekemans 2002). For testing statistical significance, 20000 permutations were used. Contribution of stepwise mutations on genetic structure, that is, whether parameter RST is better suited for the analysis of the given dataset that FST, was tested with the same programme (20000 permutations). Additionally, distance based clustering methods were used in an attempt to detect genetic structure between populations. Neighbour-joining and UPGMA trees were constructed, based on Cavalli-Sforza and Edwards chord distance DC (1967, cit. after Takezaki and Nei 1996) and Nei’s standard genetic distance DS (Nei 1972). The chord distance DC is thought to be best for the construction of tree topology, while DS and Goldstein’s (δµ)2 (Goldstein et al. 1995) are better for the estimation of branch lengths (Takezaki and Nei 1996). Goldstein’s distance (δµ)2, designed specifically for microsatellites, was not used, because no contribution of stepwise mutations was observed for our dataset (see results). Programme Populations 1.2.30 (Langella 1999) was used for the calculation of genetic distances and tree construction. The bootstrap value was set to 10000.
Isolation by distance, i.e. positive relationship between geographical and genetic distances, between sampled populations was estimated using the Mantel test implemented in Genepop 4.0 (Rousset 2008). We performed 20000 random permutations between the matrix of pair-wise genetic differences between populations calculated as FST / (1 – FST) and the matrix of the natural logarithm of geographic distance. Under isolation by distance, the values of pair-wise FST / (1 – FST) ratios are expected to increase linearly with the logarithm of distance in a two-dimensional model (Rousset 1997).
Sequential Bonferroni corrections according to Rice (1989) for testing multiple comparisons were applied where appropriate to reduce the total type I error to 0.05, 0.01 and 0.001.
Data was managed and input files in different formats prepared with MolekBase ( 151).
Genetic diversity – Genetski diverzitet
All five microsatellite loci scored were highly polymorphic, displaying a high number of alleles (from 13 to 42 per locus). Total gene diversities ranged between 0.459 on locus FEMSATL16 to 0.960 on locus M2-30 (Table 2).
High levels of genetic diversity were observed in the analysed populations (Figure 1) with mean number of alleles per population and locus between 12.8 (Razpotje) and 15.6 (Soteska). Totally 38 private alleles, i.e. alleles only present in one population, were found in the dataset, ranging from four in population Razpotje to 12 in population Rodik and from 6 on locus M2-30 to 9 on loci FEMSATL4 and 11. No