Primer Design for trnL-trnF IGS Sequences of the Quercus L. Species

The genus Quercus L., which has taxonomically problematic species, is distributed in wide geographical regions and represents the important part of forest ecosystems. Distribution in wide geographical areas is the one of the most important reasonsto increase the variations in the species, because of different climatic and geographical conditions. Furthermore, the presence of hybridization, self-incompatibility system and weak reproductive barriers between species are other important reasons to make problematic the genus. The studies based on DNA barcoding have been frequently used to determine and solve such problems. The trnL-trnF IGS sequences belonging to cpDNA is one of the most used regions to evaluate the phylogenetic relationships of the species, in addition to species identification and separation. However, it is observed that the primers used to provide trnL-trnF IGS region are universal. In other words, primer pairs devoloped for trnL-trnF IGS sequences have not been specifically designed by examination of sequences belonging to Quercus species. In this study, eight primer pair were developed from the target DNA sequencesof all Quercus species and their sequence compatibilities were tested in different species of the genus. Furthermore, in addition to length, melting temperature and GC content in primer design for trnL-trnF IGS sequences, the secondary structures tendencies of the primers such as homodimer, heterodimer and hairpin were examined in detailed. Although there are large nucleotide regions containing trnL-trnF IGS sequences for many Quercus species in the NCBI database, it was observed that the start and end regions of these sequences were not determined and were have been shared as a whole. In this study, the initial and final sequences of the trnL-trnF IGS region for Quercus species were determined and in this context, the study has also high importantance in terms of determining how many nucleotides are synthesized from the genes aroundthe relevant region in PCR amplification.