GISH Analysis of Monococcum Metaphase Chromosomes

GISH analysis of mitotic metaphase chromosomes was performed according to a previously described fluorescence in situ hybridization protocol with minor modifications[33 (link),44 (link)]. Genomic DNA was extracted from young leaves of a T. monococcum ssp. aegilopoides accession, KU-3620. Genomic DNA of KU-3620 was labeled using Biotin-Nick Translation Mix (Roche Diagnostics, Basel, Switzerland), incubated at 16°C for 24 h, and then digested with the restriction enzyme HaeIII at 37°C for 5 h. The biotin-labeled DNA was used as a probe. After chromosomes were incubated in 2× SSC (saline-sodium citrate) buffer including 70% (v/v) formamide at 80°C for 2 min for denaturation, they were hybridized with the biotin-labeled probe. The biotin-labeled probe was visualized using streptavidin-conjugated Alexa Fluor 555 (Life Technologies, Carlsbad, CA, USA). Chromosomes were counterstained with 0.1 μg/ml 4,6-diamino-2-phenylindole (DAPI). GISH signals and DAPI-stained chromosomes were captured using a fluorescence microscope (Axioskop2, Carl Zeiss, Oberkochen, Germany), and images were pseudo-colored and processed using ZEN software blue edition (Carl Zeiss).