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Background    Brachypodium distachyon  s. l. has been widely investigated across the world as a model plant for temperate cereals and biofuel grasses. However, this annual plant shows three cytotypes that have been recently recognized as three independent species, the diploids  B. distachyon  (2n = 10) and  B. stacei  (2n = 20) and their derived allotetraploid  B. hybridum  (2n = 30).    Methodology/Principal Findings  We propose a DNA barcoding approach that consists of a rapid, accurate and automatable species identification method using the standard DNA sequences of complementary plastid ( trn LF) and nuclear (ITS, GI) loci. The highly homogenous but largely divergent  B. distachyon  and  B. stacei  diploids could be easily distinguished (100% identification success) using direct  trn LF (2.4%), ITS (5.5%) or GI (3.8%) sequence divergence. By contrast,  B. hybridum  could only be unambiguously identified through the use of combined  trn LF+ITS sequences (90% of identification success) or by cloned GI sequences (96.7%) that showed 5.4% (ITS) and 4% (GI) rate divergence between the two parental sequences found in the allopolyploid.    Conclusion/Significance  Our data provide an unbiased and effective barcode to differentiate these three closely-related species from one another. This procedure overcomes the taxonomic uncertainty generated from methods based on morphology or flow cytometry identifications that have resulted in some misclassifications of the model plant and its allies. Our study also demonstrates that the allotetraploid  B. hybridum  has resulted from bi-directional crosses of  B. distachyon  and  B. stacei  plants acting either as maternal or paternal parents.

A DNA Barcoding Method to Discriminate between the Model Plant Brachypodium distachyon and Its Close Relatives B. stacei and B. hybridum (Poaceae)