DNA Barcoding In Fragaria Species

Species identification using a short DNA sequence, i.e., DNA barcoding, has been successful in animals due to the rapid mutation rate of their mitochondrial genome. In plants, several potential barcode loci have been proposed and evaluated. We chose to focus on two potential regions, the chloroplast PsbA-trnH spacer and the nuclear ribosomal internal transcribed spacer (ITS).  The objective of this study was to analyze these two regions to determine their application in barcoding Fragaria species. PsbA-trnH was successfully amplified and sequenced in 26 Fragaria accessions representing 19 species with an average sequence length of 400 bp. Fifty ITS sequences representing 19 Fragaria species were analyzed in this study. Forty-two of these ITS sequences were available from GenBank. The remaining eight ITS regions representing seven species were successfully amplified and sequenced with an average sequence length of 607 bp. Kimura two parameter (K2P) distances ranged from 0–1.9 within species, and from 0–4.4 between species. The range and average of the K2P distances between species was higher in the ITS (0-4.4%, 0.016 on average) than the PsbA-trnH (0-3.5%, 0.011 on average). The range of the within species K2P distances (0–1.9) overlapped with the between species K2P (0–4.4) which limits the use of DNA barcoding in Fragaria. The ITS neighbor joining tree clustered three tetraploids, F. tibetica Staudt & Dickore, F. corymbosa Losinsk and F. gracilis Losinsk. together. The ITS and PsbA-trnH neighbor joining trees revealed a close relationship between the fourth tetraploid used in this study, F. orientalis Losinsk, and the decaploid, F. iturupensis Staudt. The phylogenetic resolution of Fragaria species using nuclear and chloroplast genome regions has remained low. Complete chloroplast genome sequencing is now possible due to parallel sequencing technology and sample multiplexing. This technique may prove useful in identifying genomic regions that are more phylogenetically informative for Fragaria species.