A nuclear DNA barcode for eastern North American oaks and application to a study of hybridization in an Arboretum setting

DNA barcoding has proved difficult in a number of woody plant genera, including the ecologically important oak genus Quercus. In this study, we utilized restrictionsite-associated DNA sequencing (RAD-seq) to develop an economical single nucleotide polymorphism (SNP) DNA barcoding system that suffices to distinguish eight common, sympatric eastern North American white oak species. Two de novo clustering pipelines, PyRAD and Stacks, were used in combination with postclustering bioinformatic tools to generate a list of 291 potential SNPs, 80 of which were included in a barcoding toolkit that is easily implemented using MassARRAY mass spectrometry technology. As a proof-of-concept, we used the genotyping toolkit to infer potential hybridization between North American white oaks transplanted outside of their native range (Q.michauxii, Q.montana, Q muehlenbergii/Q.prinoides, and Q.stellata) into a horticultural collection surrounded by natural forests of locally native trees (Q.alba and Q.macrocarpa) in the living collection at The Morton Arboretum (Lisle, IL, USA). Phylogenetic and clustering analyses suggested low rates of hybridization between cultivated and native species, with the exception of one Q.michauxii mother tree, the acorns of which exhibited high admixture from either Q.alba or Q.stellata and Q.macrocarpa, and a hybrid between Q.stellata that appears to have backcrossed almost exclusively to Q.alba. Together, RAD-seq and MassARRAY technologies allow for efficient development and implementation of a multispecies barcode for one of the more challenging forest tree genera