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ŠUMARSKI LIST 5-6/2012 str. 38     <-- 38 -->        PDF

ash is possible, selfed seeds, which have lower genetic variability, may not survive because of inbreeding depression. Levels of selfing and seed set are similar in female and hermaphrodite trees. It is therefore unlikely that seed collection from hermaphrodite trees would result in the use of selfed seed or reduced levels of genetic diversity (FRAXIGEN 2005). Dispersal of pollen and succeeding siring of seed strongly depend on the density of the stand, the proportion of trees flowering and the landscape. Majority of common ash pollen travels over short distances, approximately 45 m (70 m in non-mast years) (FRAXIGEN 2005) to approximately 330 m between isolated patches of common ash in the barren landscape of Scotland (Bacles et al. 2005), although a proportion of pollen travels over substantial distances (Bacles and Ennos 2008). Mast years do not occur regularly and abundant flowering does not necessarily mean an abundant seed crop, especially if the weather is unsuitable for wind pollination or late frosts destroy the flowers (FRAXIGEN 2005). The fully developed seeds start to disperse by wind in the autumn. Seed dormancy usually lasts for two to six winters. Stored seed requires combined warm-cold stratification to germinate (Pliûra and Heuertz 2003).
Common ash usually regenerates naturally, but when and where natural regeneration fails, it is exceedingly important to use site adapted reproductive material of high genetic diversity to preserve natural genetic variability of the species, ensure its future adaptability and reduce the costs of artificial regeneration through successful seedling establishment, especially under changed environmental and disease conditions (Rajora and Mosseler 2001, Koskela et al. 2007). Local genotypes are assumed to be better adapted to local conditions due to natural selection, although initial results of reciprocal transplant experiments in Britain show no evidence of home site advantage for British provenances of common ash, while common ash from continental Europe is poorly adapted to British climate (Boshier and Stewart 2005). In order to ensure site adaptability, which in common and narrow-leaved ash seems to stretch over large distances (Boshier and Stewart 2005, Bogdan et al. 2007), the whole territory of Slovenia represents one provenance region delineated into four altitudinal belts for common ash (Kutnar et al. 2002). Transfers of ash reproductive material between altitudinal belts should be avoided, except in strictly defined cases (Rules on the designation... 2003). Reductions in genetic diversity (i.e. variation within populations / species that is attributable to differences in units of hereditary information) can predispose populations / species / forests to environment-related decline in health, productivity and its ability to reproduce (Rajora and Mosseler 2001, Hubert and Cottrell 2007, Hosius et al. 2006). Here common as well as low frequency and rare alleles are important because the first represent current genetic potential of the populations and the latter two latent genetic potential (Rajora and Mosseler 2001, Hattemer 1995). Forestry practices such as sylvicultural system, regeneration method, thinning as well as seed harvesting and processing can greatly affect genetic diversity within the stand. Collection of seed from a small number of trees spaced closely together, where the bulk of seed was collected from only a part of all selected trees can substantially reduce genetic diversity of the seed lot, which can be further reduced during careless seed processing and culling of young plants in the nursery (Hosius et al. 2006, Schmidt 2000).
When talking about genetic diversity, sample size is very important. Mean square error estimates of resampled data from a common ash stand in France indicated that more than 300 individuals are necessary for accurate measures of allelic richness (number of alleles, number of effective alleles) while estimation of expected heterozygosity (gene diversity) requires smaller samples (< 30). Intermediate sample sizes are needed for accurate estimation of the inbreeding coefficient (Miyamoto  et al. 2008).
We have used nuclear microsatellites to investigate patterns of neutral genetic diversity and population genetic structure of common ash in five populations from Slovenia. In particular we were interested in the following questions: (1) Are the two analysed approved seed stands better suited for collection of forest reproductive material from a genetic perspective than non-approved stands? (2) Is the existing one provenance region for the whole country sufficient for common ash? We are however not addressing the question connected with climate change, in which the principle ‘local is best’ based on assumption that natural selection has optimised populations to their local environment, endorsed by the Helsinki guidelines (MCPFE 1993), European (1999/105/EC) and Slovenian (Rules on the designation... 2003) legislation, is questioned with respect to the oncoming climate change.
Materials and Methods
Materijal i metode
Plant material, DNA isolation and microsatellite analysis – Biljni materijal, izolacija DNA i analiza mikrosatelita
Twigs of 148 Fraxinus excelsior trees from five populations were sampled (Table 1). Genomic DNA was extracted from approximately 15 × 2 mm large strip of cambium using 2 % CTAB extraction buffer as described by Doyle and Doyle (1990). Microsatellite analysis was performed with five primer pairs (FEMSATEL4, FEMSATEL11, FEMSATEL16, FEMSATEL19, M2-30) developed by Lefort et al. (1999) and Brachet et al. (1999). PCR reactions were performed in a GeneAmp® 9700 thermocycler in a reaction mix containing 2 mm MgCl2, 0.2 U Taq polymerase, 1× PCR buffer, 0.4 mm