Primers for 52 polymorphic regions in the Quercus rubra chloroplast, 47 of which amplify across 11 tracheophyte clades

Abstract

Postglacial migration studies in Quercus rubra L. (northern red oak) are hampered by low levels of population differentiation in the widely used universal chloroplast primers. We sequenced the large single copy (LSC) regions of the Q. rubra and Quercus ellipsoidalis chloroplasts to enable us to query additional regions for future studies on migration and speciation. Using 454 sequencing of long-range PCR amplicons and Sanger sequencing for gap closure, we report 65 coding sequences from Q. rubra and 59 from Q. ellipsoidalis. Comparison of our de novo assembly of the LSC region sequence for Q. rubra to Q. rubra chloroplast sequence (NCBI Reference Sequence: NC_020152.1) from a different tree revealed 106 polymorphisms, all within intergenic regions, that can serve as tools for postglacial migration studies and taxonomic studies within the Lobatae. Sequence alignment for the 59 complete coding regions in common for theQ. rubrachloroplast reference sequence, our Q. rubra sequence and our Q. ellipsoidalis sequence revealed no sequence polymorphisms and no indels. We also report the 52 primer pairs we used for gap closure, including 53 new primer pairs not previously reported. We tested these 52 primer pairs against 11 species representing the Tracheophyta and detected 47 that produced amplicons in all 11 species. The new universal primers we have identified provide additional tools for resolving the taxonomic relationships among the congeneric taxa of forest trees in the temperate and subtropical forests of the Northern Hemisphere.