Figure 1.
Figure 2.
PCR protocol temperature regime for two separate PCR reactions (mix 1 and mix 2)_
| Protocol | |||
|---|---|---|---|
| Temperature (°C) | Duration (min:s) | Number of cycles | |
| Enzyme activation | 95 | 5:00 | |
| Denaturation | 94 | 0:40 | |
| Annealing | 52 | 0:40 | 35 |
| Elongation | 72 | 0:30 | |
| Final elongation | 72 | 4:00 | |
Results of F statistical analysis_
| Locus | FIS | FIT | FST |
|---|---|---|---|
| CM007 | -0.111 | -0.086 | 0.022 |
| CM008 | 0.154 | 0.201 | 0.056 |
| CM010 | 0.161 | 0.191 | 0.036 |
| CM026 | 0.146 | 0.189 | 0.051 |
| CM031 | 0.059 | 0.083 | 0.025 |
| CM037 | -0.077 | -0.052 | 0.023 |
| CM039 | -0.029 | 0.012 | 0.040 |
| CM043 | 0.150 | 0.224 | 0.086 |
| Average | 0.057 | 0.095 | 0.042 |
Proportion of components used in PCR reaction mix 1 and mix 2_
| Mix 1 | Mix 2 | ||
|---|---|---|---|
| Components | Reaction concentration | Components | Reaction concentration |
| CM007 | 0.1 μM | CM031 | 0.2 μM |
| CM008 | 0.2 μM | CM037 | 0.2 μM |
| CM010 | 0.1 μM | CM039 | 0.2 μM |
| CM026 | 0.1 μM | CM043 | 0.2 μM |
| dNTP | 0.2 mM | dNTP | 0.2 mM |
| PCR buffer | 1× | PCR buffer | 1× |
| MgCl2 | 2.5 mM | MgCl2 | 2.5 mM |
| Taq pol. | 0.2 U · μL-1 | Taq pol. | 0.2 U · μL-1 |
| DNA | 10 ng | DNA | 10 ng |
| ddH2O | to 15 μL | ddH2O | to 15 μL |
Deviation of eight examined SSR loci from HW equilibrium in the total set samples_
| Population | Locus | SS | χ2 | p-value | Significance |
|---|---|---|---|---|---|
| Drvar | CM007 | 45 | 63.942 | 0.033 | * |
| Drvar | CM008 | 36 | 71.652 | 0.000 | *** |
| Drvar | CM010 | 36 | 76.893 | 0.000 | *** |
| Drvar | CM026 | 21 | 14.129 | 0.864 | ns |
| Drvar | CM031 | 55 | 61.856 | 0.245 | ns |
| Drvar | CM037 | 36 | 27.500 | 0.845 | ns |
| Drvar | CM039 | 28 | 58.890 | 0.001 | *** |
| Drvar | CM043 | 15 | 14.860 | 0.462 | ns |
| Mostar | CM007 | 78 | 90.510 | 0.157 | ns |
| Mostar | CM008 | 55 | 75.422 | 0.035 | * |
| Mostar | CM010 | 91 | 104.822 | 0.153 | ns |
| Mostar | CM026 | 21 | 22.016 | 0.399 | ns |
| Mostar | CM031 | 28 | 28.657 | 0.430 | ns |
| Mostar | CM037 | 55 | 56.788 | 0.408 | ns |
| Mostar | CM039 | 28 | 69.540 | 0.000 | *** |
| Mostar | CM043 | 21 | 18.345 | 0.627 | ns |
| Zenica | CM007 | 45 | 48.200 | 0.345 | ns |
| Zenica | CM008 | 28 | 10.674 | 0.999 | ns |
| Zenica | CM010 | 45 | 55.142 | 0.143 | ns |
| Zenica | CM026 | 10 | 15.985 | 0.100 | ns |
| Zenica | CM031 | 55 | 41.147 | 0.917 | ns |
| Zenica | CM037 | 21 | 15.396 | 0.803 | ns |
| Zenica | CM039 | 15 | 78.395 | 0.000 | *** |
| Zenica | CM043 | 6 | 5.235 | 0.514 | ns |
Parameters of heterozygosity_
| Populations | Locus | Na | Ne | R | P (R) | Ho | He | F |
|---|---|---|---|---|---|---|---|---|
| Drvar | CM007 | 10.000 | 7.339 | 0.735 | 0.1979 | 0.950 | 0.864 | -0.100 |
| CM008 | 9.000 | 5.517 | 0.614 | 0.1311 | 0.550 | 0.819 | 0.328 | |
| CM010 | 9.000 | 6.723 | 0.746 | 0.2046 | 0.550 | 0.851 | 0.354 | |
| CM026 | 7.000 | 3.846 | 0.549 | 0.1 | 0.650 | 0.740 | 0.122 | |
| CM031 | 11.000 | 4.938 | 0.45 | 0.059 | 0.600 | 0.798 | 0.248 | |
| CM037 | 9.000 | 4.348 | 0.483 | 0.0717 | 0.800 | 0.770 | -0.039 | |
| CM039 | 8.000 | 5.298 | 0.661 | 0.1556 | 0.850 | 0.811 | -0.048 | |
| CM043 | 6.000 | 4.145 | 0.692 | 0.1727 | 0.700 | 0.759 | 0.077 | |
| Mostar | CM007 | 13.000 | 7.339 | 0.566 | 0.1078 | 0.950 | 0.864 | -0.100 |
| CM008 | 11.000 | 8.000 | 0.727 | 0.1931 | 0.750 | 0.875 | 0.143 | |
| CM010 | 14.000 | 9.877 | 0.707 | 0.1813 | 0.850 | 0.899 | 0.054 | |
| CM026 | 7.000 | 4.571 | 0.652 | 0.1508 | 0.750 | 0.781 | 0.040 | |
| CM031 | 8.000 | 5.063 | 0.634 | 0.1413 | 0.800 | 0.803 | 0.003 | |
| CM037 | 11.000 | 5.594 | 0.508 | 0.082 | 0.850 | 0.821 | -0.035 | |
| CM039 | 8.000 | 5.298 | 0.661 | 0.1556 | 0.700 | 0.811 | 0.137 | |
| CM043 | 7.000 | 4.651 | 0.664 | 0.1572 | 0.650 | 0.785 | 0.172 | |
| Zenica | CM007 | 10.000 | 8.602 | 0.862 | 0.2857 | 1.000 | 0.884 | -0.132 |
| CM008 | 8.000 | 3.687 | 0.461 | 0.0631 | 0.750 | 0.729 | -0.029 | |
| CM010 | 10.000 | 7.843 | 0.787 | 0.2309 | 0.800 | 0.873 | 0.083 | |
| CM026 | 5.000 | 4.188 | 0.837 | 0.2663 | 0.550 | 0.761 | 0.278 | |
| CM031 | 11.000 | 4.790 | 0.435 | 0.0535 | 0.850 | 0.791 | -0.074 | |
| CM037 | 7.000 | 4.469 | 0.638 | 0.1434 | 0.900 | 0.776 | -0.159 | |
| CM039 | 6.000 | 3.448 | 0.575 | 0.112 | 0.850 | 0.710 | -0.197 | |
| CM043 | 4.000 | 2.067 | 0.516 | 0.0854 | 0.400 | 0.516 | 0.225 | |
| Average (Drvar) | 8.625 | 5.269 | 0.584 | 0.1163 | 0.706 | 0.801 | 0.118 | |
| Average (Mostar) | 9.875 | 6.299 | 0.595 | 0.1217 | 0.788 | 0.830 | 0.052 | |
| Average (Zenica) | 7.625 | 4.887 | 0.535 | 0.0937 | 0.763 | 0.755 | -0.001 | |
| Average (Total) | 8.708 | 5.485 | 0.561 | 0.1055 | 0.752 | 0.795 | 0.056 |
SSR markers and characteristics_
| SSR markers | Primer sequence (5′ → 3′) | A repetitive pattern |
|---|---|---|
| CM007 | R:tccagggaatgttcggtagattag L:gtttaggtgtgagtgcagatgg | (GT)24 |
| CM008 | L:tcgttaatgtgaaattggaacg R:caccgtacacgcaaagtcc | (GT)11 |
| CM010 | L:gctagcagaagcacagttagcc R:tccaacatgtaaaacctagatgc | (CA)12 |
| CM026 | L:gaattcatgtaatgttgttgtctgc R:cctgcatataattcaggtaaagagc | (CA)14 |
| CM031 | L:taccctctcttgctctttgtcc R:aaacaatcaaacccaaacaacc | (AG)26(TG)13 |
| CM037 | L:aacacagagaaacacgtgcaa R:tggagatctttgaagaacagga | (TG)20 |
| CM039 | L:gggtattgtaatcaatgtaaaaccaa R:tcacaccaccagcaatcact | (GT)18 |
| CM043 | L:gtccacacctgttgttcagc R:ggttgcaatgctttcttgg | (TG)16(TA)5 |
AMOVA_
| Source | df | SS | MS | Estimated Var. | % |
|---|---|---|---|---|---|
| Among Pops | 2 | 16.650 | 8.325 | 0.120 | 3 |
| Among Indiv. | 57 | 201.300 | 3.532 | 0.262 | 8 |
| Within Indiv. | 60 | 180.500 | 3.008 | 3.008 | 89 |
| Total | 119 | 398.450 | 3.390 | 100 |