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Breeding of agricultural crops adapted to climate change and resistant to diseases and pests is hindered by a limited gene pool because of domestication and thousands of years of human selection. One way to increase genetic variation is chromosome-mediated gene transfer from wild relatives by cross hybridization. In the case of wheat ( Triticum aestivum ), the species of genus  Aegilops  are a particularly attractive source of new genes and alleles. However, during the evolution of the  Aegilops  and  Triticum  genera, diversification of the D-genome lineage resulted in the formation of diploid C, M, and U genomes of  Aegilops . The extent of structural genome alterations, which accompanied their evolution and speciation, and the shortage of molecular tools to detect  Aegilops  chromatin hamper gene transfer into wheat. To investigate the chromosome structure and help develop molecular markers with a known physical position that could improve the efficiency of the selection of desired introgressions, we developed single-gene fluorescence  in situ  hybridization (FISH) maps for M- and U-genome progenitors,  Aegilops comosa  and  Aegilops umbellulata , respectively. Forty-three ortholog genes were located on 47 loci in  Ae. comosa  and on 52 loci in  Ae. umbellulata  using wheat cDNA probes. The results obtained showed that M-genome chromosomes preserved collinearity with those of wheat, excluding 2 and 6M containing an intrachromosomal rearrangement and paracentric inversion of 6ML, respectively. While  Ae. umbellulata  chromosomes 1, 3, and 5U maintained collinearity with wheat, structural reorganizations in 2, 4, 6, and 7U suggested a similarity with the C genome of  Aegilops markgrafii . To develop molecular markers with exact physical positions on chromosomes of  Aegilops , the single-gene FISH data were validated  in silico  using DNA sequence assemblies from flow-sorted M- and U-genome chromosomes. The sequence similarity search of cDNA sequences confirmed 44 out of the 47 single-gene loci in  Ae. comosa  and 40 of the 52 map positions in  Ae. umbellulata . Polymorphic regions, thus, identified enabled the development of molecular markers, which were PCR validated using wheat- Aegilops  disomic chromosome addition lines. The single-gene FISH-based approach allowed the development of PCR markers specific for cytogenetically mapped positions on  Aegilops  chromosomes, substituting as yet unavailable segregating map. The new knowledge and resources will support the efforts for the introgression of  Aegilops  genes into wheat and their cloning.

Development of DNA Markers From Physically Mapped Loci in Aegilops comosa and Aegilops umbellulata Using Single-Gene FISH and Chromosome Sequences