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These microsatellite markers are among those selected to genotype European oak species in recent studies [26][17][31]. Several other markers were tested e.

Polymerase chain reactions PCRs were conducted in a Corbett thermal cycler.

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The forward primer was fluorescently labelled with Beckman dyes: D2, D3 or D4. The primer pairs were combined into three multiplex reactions. The first set comprised four markers, and dye type and primer concentrations μM are given in brackets: QrZAG D4, 0.

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The PCR profile was as follows: 3 min of denaturation at 94 °C followed by 31 cycles of 50 s denaturation at 94 °C, a 40 s annealing step at 52 °C, a 1 min 20 s elongation step at 70 °C and a final extension step at 70 °C for 12 min. The products were then analyzed using Fragment Analysis Software using default parameters and PA ver1 dye correction.

Study site and sampling

First, we used the 10 microsatellite markers on a group of eight oak populations distributed across Romania Tab. S1 in Appendix 1. No evidence of large allele drop-outs or scoring of stutter peaks fat matt s burn found in the data set.

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However, simulations showed that at such low frequencies, these null alleles do not affect the overall outcome of the genetic assignment [5] or the robustness of SGS results [36]. Finally, we used the same set of genetic markers to genotype the oak individuals sampled at the Bejan oak mixed forest. Simulations were run for steps following a burn-in period of 50 steps, considering values of K number of clusters from one to 10, with 10 replications for each value of K.

The analysis was performed using admixture, correlated allele frequencies and no prior information regarding sampling location or morphological species.

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For each individual the program identifies the fraction of the genome that belongs to each one of the clusters. First, we ran STRUCTURE for the set of eight external pure populations individualswhich consisted of two morphologically pure populations for each of the four species found in the mixed forest of Bejan.

The rate of change in the log likelihood between successive K values [16] was also estimated. For the classification of individuals into purebred and hybrid classes, we followed the procedure described in a previous study on four French oak species with 10 microsatellite markers [26].

Genotyping

Each individual was considered to be a purebred when its q value admixture coefficient was larger than 0. When an individual had a q value lower than 0. Individuals with q values between 0. Spatial genetic structure analysis To study the spatial distribution of multilocus genotypes within both species and the total population, we computed the correlation coefficient r [41] using the program GENALEX 6.

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The maximum pairwise distance between individuals sampled within the mixed forest varied between Because the sample size for each species varied considerably and the number of pairs within each distance class should be at least [19]we set the distance intervals to 25 m.

To compare the results between species, we also chose to conduct the analyses for 10 distance intervals between 0 and m. The nonparametric heterogeneity test of Smouse et al.

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The number of bootstrap resamplings was set to The significance of the slope of the regression analysis was computed after 10 permutations. Sp confidence intervals were obtained as described in [49].

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S1 - Appendix 1. We then ran the software for the whole sample set, including the trees sampled in the mixed forest. The overall percentage of putative hybrids in the mixed forest was high The highest proportions of hybrids, The most fat matt s burn pure species was Q.

The highest hybridization rate was observed between Q. Populations are separated by a thin black line. Populations consist of oaks sampled in Bejan mixed forest, which are grouped to the species they most closely resemble: Q. Regardless of the species, oak individuals in the shortest distance interval appeared to be more genetically related than expected based on a random distribution.

The correlation coefficient was significantly positive for fat matt s burn first two distance classes up to 50 m only for Q. The SGS patterns were characterized by a peak r value at the first distance class, followed by a more or less steady decrease for the remaining distance intervals Fig. Overall, the heterogeneity test of SGS between each pair of species was not significantly different, nor for each distance class data not shown.

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  • He shall pour out the rest of the blood at the base of the altar used for burning sacrifices, which is at the entrance of the Tent.
  • Which shows that not the fat only, but the inwards and the kidneys, were burnt also; so Maimonides says, that the priest salted the parts, and burned them upon the altar; and the priests might not have the breast and shoulder which were what belonged to them until the parts were burnt: it is the food of the offering made by fire; which the Lord ate of, or accepted of: for a sweet savour; as a type of the sweet smelling sacrifice of Christ, with which he is well pleased: all the fat is the Lord's; that is, all that was upon the parts mentioned in the several sacrifices of peace offerings, which was to be taken off and burnt: though the Jewish writers understand it of all fat in general, and so interpret the law that follows.

On x-axis are 25 m distance intervals. However, the SGS was no longer significant for Q. The statistical tests for heterogeneity between pairs of species were also not significant data not shown.

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The value of the Sp statistic was highest for Q. The value of bF was significantly different from zero in all four species Tab. After hybrids removal, Sp statistic values were no longer significantly different between species. Interestingly, Q. After the removal of hybrids, Sp statistic value only increased for Q. This species showed the highest proportion of hybrids SGS Analysis.

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