Taxonomic and ecological discrimination of Fagaceae species based on internal transcribed spacer polymerase chain reaction–restriction fragment length polymorphism

The internal transcribed spacer (ITS) of ribosomal DNA has been used to confirm taxonomic classifications and define phylogenies in several plant species following sequencing or polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) techniques. In this study, co-dominant ITS PCR–RFLP molecular markers were produced in 30 Fagaceae individuals belonging to the Castanea, Fagus and Quercus genera in order to assess the potential of this technique for taxonomic discrimination and determination of phylogenies. The complete ITS region (ITS1-5.8S rRNA-ITS2) was amplified in most of the Fagaceae individuals as a single fragment of ∼700 bp. The ITS amplified products were digested with nine restriction enzymes, but only four (HaeIII, HpaII, TaqI and Sau96I) produced polymorphic/discriminative patterns. The total expected heterozygosity (H(E)) was 20.31 % and the gene diversity (I), 32.97 %. The ITS polymorphism was higher within the Quercus genus (85.3 %). The ITS PCR–RFLP markers clustered the Fagaceae species according to genus or infrageneric group (in the case of Quercus sp. individuals). Five oaks did not cluster in line with the adopted infrageneric classification, but three of these were grouped according to their actual ecological distributions. The ITS PCR–RFLP markers indicated their potential for phylogenetic studies since all Fagaceae individuals were discriminated according to genus, and most of the oaks were clustered according to infrageneric group or ecological area.