2.2. Species identification
Species identification and ploidy level characterization were conducted for all of the accessions that were used in different experiments. Two complementary identification methods were used.Nuclei were isolated from young leaves (40 mg) by chopping with a razor blade in ice-cold LB01 buffer [27–29]. Nuclei were then sep-arated using cotton squares that were placed on the top of 10-mLglass columns and total DNA was stained with propidium iodide[30]. Flow cytometry (FACS Calibur, BD Biosciences) was used todetermine DNA content and confer ploidy levels [31], in compar-ison with two previously known accessions Bd-21 (Brachypodiumdistachyon) and Hawalid (Brachypodium stacei) [32].DNA was extracted from fresh leaves of the same accessions andPCR was carried out under known conditions with microsatellitemarker XALB165 [33]. Amplification products from each accession1All