Bivariate flow cytometry of wheat-Aegilops addition lines facilitate dissecting the genomes of Ae. geniculata and Ae. biuncialis into individual chromosomes

Said M., Molnár M., Farkas A., Ivanizs L., Gaál E., Capal P., Doležel J
Book of Abstracts - 11th International Conference Analytical Cytometry - October 2-5 - Ostrava - Czech Republic : , 2021

Abstract: Allotetraploid goatgrasses Aegilops. geniculata and Ae. biuncialis are attractive sources of beneficial traits for wheat improvement. However, transfer of favorable traits from these species into wheat has been hampered by poor knowledge of their genomes and scarcity of molecular tools for efficient screening of hybrid progenies. Purification of individual chromosomes by flow cytometric sorting would make it possible to sequence the complex genomes chromosome by chromosome and develop chromosome-specific DNA markers. However, the ability to sort individual chromosomes relies on a possibility to discriminate them. In this work, we performed bivariate flow karyotyping of chromosome suspensions prepared from both species. The chromosomes were labeled with FITC-conjugated probe for (GAA)7 microsatellite and their DNA was stained by DAPI. Flow karyotypes of Ae. biuncialis (Figure 1) and Ae. geniculata (Figure 2) comprised nine and fourteen chromosome populations, representing one to five and three to five chromosomes, respectively. In order to purify single chromosomes, bivariate analysis of DAPI vs. FITC fluorescence was performed with suspensions of chromosomes prepared from wheat-Ae. biuncialis and wheat-Ae. geniculata chromosome addition lines. Individual chromosomes of the two species could be sorted at purities ranging from 74.5 to 96.6% and from 87.8 to 97.7%, respectively. Flow-sorted chromosomes will streamline genome analysis of Ae. biuncialis and Ae. geniculata, facilitate gene cloning and enable development of molecular tools to support alien introgression breeding of wheat. Acknowledgements: Czech Ministry of Education, Youth and Sports and the European Regional Development Fund (ED0007/01/01) and OTKA K135057.
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IEB authors: Petr Cápal, Jaroslav Doležel, Istvan Molnar, Mahmoud Said