An rbcL reference library to aid in the identification of plant species mixtures by DNA metabarcoding1

This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY-NC-SA 4.0), which permits unrestricted noncommercial use and redistribution provided that the original author and source are credited and the new work is distributed under the same license as the original. DNA metabarcoding, the use of high-throughput sequencing methods to simultaneously DNA barcode all species in a mixed sample, can been used to address a variety of questions in ecology , biosecurity, and forensics, among other fields (Cristescu, 2014; Bell et al., 2016a, 2016b). These methods enable taxo-nomic identifications of plant samples in which diagnostic characters are largely absent (e.g., roots in a soil sample, seeds from seed traps, or pollen), or for which few experts know the diagnostic characters. DNA metabarcoding has streamlined identifications of pollen for allergen monitoring (Kraaijeveld et al. The analysis of fragments of plant material in herbivore guts enables diet analysis (Valentini et al., 2009; Pompanon et al., 2012). DNA metabarcoding can also be used to determine the species composition of dietary supplements (Cheng et al., 2014) and foods (Hawkins et al., 2015) leading to improved health and safety. However, accurate determination of species by DNA me-tabarcoding is dependent on a comprehensive and accurate reference library of DNA sequences of a standard genetic marker, as well as an appropriate bioinformatics pipeline. For plants, the Consortium for the Barcode of Life (CBOL) Plant Working group recommended the plastid DNA (ptDNA) genes rbcL and matK as standard DNA barcode markers, based on the availability of universal primers and the high level of taxonomic resolution (CBOL Plant Working Group, 2009; Hollingsworth et al., 2009). Recent studies using the standard rbcL+matK barcode for floras of moderate phylogenetic dispersion have shown that up to 92% of the species can be distinguished (Kress et al., 2009; Burgess et al., 2011). Amplicon fragment size is important in high-throughput sequencing (HTS) DNA metabarcoding, as the read lengths in many platforms are currently limited (e.g., ~600-bp paired-end reads on the Illumina long amplicon length generated by standard matK primers poses a technical limitation for these sequencing methods. In addition to ptDNA markers, the nuclear ribosomal spacer ITS2 has been shown to be an effective DNA barcoding marker, with 92.7% successful identifications in 6600 samples (Chen et al., 2010). A bioinformatics pipeline has been published for the identification of species mixtures using ITS2 (Sickel et al., 2015). This bioinformatics …