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 separated using cotton squares that were placed on the top of 10-mL
glass columns and total DNA was stained with propidium iodide
[30]. Flow cytometry (FACS Calibur, BD Biosciences) was used to
determine DNA content and confer ploidy levels [31], in comparison with two previously known accessions Bd-21 (Brachypodium
distachyon) and Hawalid (Brachypodium stacei) [32].
DNA was extracted from fresh leaves of the same accessions and
PCR was carried out under known conditions with microsatellite
marker XALB165 [33]. Amplification products from each accession