Chromosome Sorting and Characterization of Aegilops Species

The preparation of mitotic metaphase chromosome suspensions of Ae. comosa MvGB1039 and Ae. umbellulata AE740/03 was carried out as described by Vrána et al. (2000 (link)) and Kubaláková et al. (2005 (link)). Prior to the flow cytometric analysis, the chromosomes were labeled by fluorescence in situ hybridization in suspension (FISHIS) using 5′-FITC-GAA₇-FITC-3′ oligonucleotides (Sigma, Saint Louis, MO, United States) according to Giorgi et al. (2013 (link)) and stained by DAPI (4′,6-diamidino 2-phenylindole) at 2 μg/ml. Chromosome analysis and sorting were carried out using a FACSAria II SORP flow cytometer and sorter (Becton Dickinson Immunocytometry Systems, San José, CA, United States) as described by Molnár et al. (2016 (link)) and Said et al. (2019 (link)). Bivariate flow karyotypes FITC vs. DAPI fluorescence were acquired for each sample and two batches of 25,000–76,000 copies of each chromosome were sorted into PCR tubes containing 40 μl sterile deionized water. The chromosome content of the flow-sorted fractions was determined by FISH on chromosomes sorted onto a microscopic slide using the probes for pSc119.2, Afa family repeat, and 45S rDNA according to Molnár et al. (2016 (link)). The chromosomes were classified following the karyotype described by Parisod and Badaeva (2020 (link)).