Genetic variation and differentiation within a natural community of five oak species (Quercus spp.).

Chloroplast DNA and two categories of nuclear markers - isozymes and microsatellites - were used to examine a very rich natural community of oaks (Quercus spp.) situated in west-central Romania. The community consists of five oak species: Q. robur, Q. petraea, Q. pubescens, and Q. frainetto - that are closely related -, and Q. cerris. A total of five chloroplast haplotypes was identified. Q. cerris was fixed for a single haplotype. The other four species shared the two most common haplotypes. One haplotype was confined to Q. robur and a very rare one was restricted to Q. petraea. Both types of nuclear markers revealed a larger genetic variation for Q. pubescens and Q. petraea than for Q. frainetto and Q. robur, although the differences between species are in most cases not significant. At the nuclear level, Q. cerris could be clearly separated from the other four oak species confirming the taxonomic classification. Regardless of the estimate used, the levels of polymorphism revealed by microsatellites were much higher than those based on isozymes. For the four closely related species the overall genetic differentiation was significant at both categories of nuclear markers. Several loci, such as Acp-C for isozymes, and ssrQpZAG36 and ssrQrZAG96 for microsatellites were very useful to discriminate among species. However, the level of differentiation varied markedly between pairs of species. The genetic affinities among the species may reflect different phylogenetic distances and/or different rates of recurrent gene flow at this site.

Novel SNP development and analysis at a NADP+ -specific IDH enzyme gene in a four species mixed oak forest.

Closely related Quercus species generally exhibit low levels of genetic differentiation despite their ecological and morphological differences. However, at a few so-called 'outlier' loci they seem to remain genetically distinct. Isocitrate dehydrogenases (IDH) are key enzymes involved in the metabolic pathway of the citrate cycle. IDH has also been characterised as an 'outlier' marker, significantly differentiating the closely related Q. robur and Q. petraea with the isozyme technique. This ability to differentiate the species was tested here at molecular level: 13 single nucleotide polymorphism (SNP) markers were identified and developed within a NADP(+) -specific IDH gene in Quercus spp. and applied as molecular markers in a four species mixed oak forest in eastern Europe, where Q. robur, Q. petraea, Q. pubescens and Q. frainetto naturally co-exist. From the 13 developed SNPs, three groups were formed: non-synonymous, synonymous and non-coding SNPs. The levels of total gene diversity were moderate for all species investigated. The non-synonymous SNPs showed lower levels of gene diversity. Overall, the four closely related Quercus spp. were significantly differentiated (except Q. petraea with Q. frainetto). Analysis of non-random association of alleles revealed no clear physical clustering of the SNP sites in significant linkage disequilibrium (LD). However, separate LD analysis for each species showed a lower number of sites in significant LD for Q. robur than for the other species, possibly reflecting the history of the species in this specific geographical site and less efficient recombination effect due to the larger effective population size of Q. robur. Eleven statistically significant associations were found between seven SNPs and morphological traits that are commonly used to differentiate oak species.