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Premise of the study:  DNA metabarcoding has broad‐ranging applications in ecology, aerobiology, biosecurity, and forensics. A bioinformatics pipeline has recently been published for identification using a comprehensive database of ITS2, one of the common plant DNA barcoding markers. There is, however, no corresponding database for  rbcL , the other primary marker used in plants.    Methods:  Using publicly available data, we compiled a reference library of  rbcL  sequences and trained databases for use with UTAX and RDP classifier algorithms. We used this reference library, along with the existing bioinformatics pipeline and ITS2 reference library, to identify species in an artificial mixture of nine species of pollen. We have made this database publicly available in multiple formats, to allow use with multiple bioinformatics pipelines, now and in the future.    Results:  Using the  rbcL  database, in addition to the ITS2 database, we succeeded in making species‐level identifications for eight species and a family‐level identification of the ninth species. This is an improvement on ITS2 sequence alone.    Discussion:  The reference library described here will assist with identification of plant species using  rbcL . By making another gene region available for standard barcoding, this will increase the resolution and accuracy of identifications.

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