Dissecting the genomes of Aegilops biuncialis and Ae. geniculata by chromosome flow sorting

Said M., Cápal P., Farkas A., Gaál E., Ivanizs L., Friebe B., Doležel J., Molnár I.
12th International Conference Analytical Cytometry : , 2023

Abstract: Breeding of wheat adapted to new climatic conditions and resistant to diseases and pests is hindered by a limited gene pool due to domestication and thousands of years of human selection. Annual goatgrasses (Aegilops spp.) with M and U genomes are potential sources of new gene variants. Development of wheat-Aegilops introgression lines can be facilitated by the knowledge of DNA sequences of U and M chromosomes. Aegilops chromosomes purified by flow cytometric sorting provide an attractive opportunity to sequence the big and complex Aegilops genomes. The present study extends the potential of chromosome genomics to allotetraploid Ae. biuncialis and Ae. geniculata by dissecting their M and U genomes into individual chromosomes. Hybridization of FITC-conjugated GAA oligonucleotide probe to chromosomes suspensions of the two species allowed the application of bivariate flow karyotyping and sorting some chromosomes. Bivariate flow karyotype FITC vs. DAPI of Ae. biuncialis consisted of nine chromosome-populations, but their chromosome content determined by microscopic analysis of flow sorted chromosomes indicated that only 7Mb and 1Ub could be sorted at high purity. In case of Ae. geniculata, fourteen chromosome-populations were discriminated allowing the separation of nine chromosomes (1Mg, 3Mg, 5Mg, 6Mg, 7Mg, 1Ug, 3Ug, 6Ug and 7Ug) out of the 14. To sort the remaining chromosomes, a partial set of wheat-Ae. biuncialis and a whole set of wheat-Ae. geniculata chromosome addition lines were also flow karyotyped revealing clear separation of the GAA-rich Aegilops chromosomes from GAA-poor A- and D-genome chromosomes of wheat. All of the alien chromosomes represented by individual addition lines could be isolated at purities ranging from 74.5 to 96.6% and from 87.8 to 97.7%, respectively. Chromosome-specific genomic resources will facilitate gene cloning and development of molecular tools to support alien introgression breeding of wheat. Acknowledgments The authors thank Dr. John Raupp from the Kansas State University, Manhattan, Kansas, USA for helping with providing the seeds of wheat cv. CS and CS-Ae. geniculata accession TA2899 chromosome addition lines 1Mg–7Mg and 1Ug–7Ug. We appreciate technical assistance of Zdeňka Dubská, Romana Šperková, and Jitka Weiserová. Funding This work has been supported by the ERDF project ‘Plants as a tool for sustainable global development’ (No. CZ.02.1.01/0.0/0.0/16_019/0000827), the Hungarian National Research, Development and Innovation Office (K135057, TKP2021-NKTA-06, and 2019-2.1.11-TÉT-2019-00074), the Marie Curie Fellowship Grant award ‘AEGILWHEAT’ (H2020-MSCA-IF-2016-746253), and the ELIXIR-CZ project (LM2015047), a component of the international ELIXIR infrastructure that is part of the project “e-Infrastruktura CZ” (LM2018140) within the program Projects of Large Research, Development and Innovations Infrastructures.
IEB authors: Petr Cápal, Jaroslav Doležel, Istvan Molnar, Mahmoud Said