Abstract
Species identification of oaks (Quercus) is always a challenge because many species exhibit variable phenotypes that overlap with other species. Oaks are notorious for interspecific hybridization and introgression, and complex speciation patterns involving incomplete lineage sorting. Therefore, accurately identifying Quercus species barcodes has been unsuccessful. In this study, we used chloroplast genome sequence data to identify molecular markers for oak species identification. Using next generation sequencing methods, we sequenced 14 chloroplast genomes of Quercus species in this study and added 10 additional chloroplast genome sequences from GenBank to develop a DNA barcode for oaks. Chloroplast genome sequence divergence was low. We identified four mutation hotspots as candidate Quercus DNA barcodes; two intergenic regions (matK-trnK-rps16 and trnR-atpA) were located in the large single copy region, and two coding regions (ndhF and ycf1b) were located in the small single copy region. The standard plant DNA barcode (rbcL and matK) had lower variability than that of the newly identified markers. Our data provide complete chloroplast genome sequences that improve the phylogenetic resolution and species level discrimination of Quercus. This study demonstrates that the complete chloroplast genome can substantially increase species discriminatory power and resolve phylogenetic relationships in plants.
Highlights
DNA barcoding has recently emerged as a new molecular tool for species identification [1]
At the third DNA barcode conference held in Mexico City in 2009, the majority of the Consortium for the Barcode of Life (CBOL) Plant Working Group preferred to recommend a core-barcode combination consisting of portions of two plastid coding regions, rbcL and matK, which are supplemented with additional markers as required
To validate the accuracy of the assembled chloroplast genome, we carried out Sanger sequencing of PCR amplicons spanning the junction regions (LSC/IRA, large single copy (LSC)/IRB, small single copy (SSC)/IRA, and SSC/IRB)
Summary
DNA barcoding has recently emerged as a new molecular tool for species identification [1]. A DNA barcode is a short, standardized DNA region normally employed for species identification. The mitochondrial gene cytochrome oxidase 1 (COI) is an effective and reliable standard animal DNA barcode for species identification [1]. Proposed barcode segments exist primarily in chloroplast genomes that are relatively stable, single-copy, and easy to amplify. These proposed barcodes are matK, rbcL, ropC1, and rpoB in the coding region, and atpF-H, trnL-F, trnH-psbA, and psbK-I in the non-coding region [2]. Increasing numbers of studies show that core-barcodes remain problematic, especially in recently diverged and rapidly radiated taxa [4,5,6]
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