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

Abstract

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 (<i>Triticum aestivum</i>), the species of genus <i>Aegilops</i> are a particularly attractive source of new genes and alleles. However, during the evolution of the <i>Aegilops</i> and <i>Triticum</i> genera, diversification of the D-genome lineage resulted in the formation of diploid C, M, and U genomes of <i>Aegilops</i>. The extent of structural genome alterations, which accompanied their evolution and speciation, and the shortage of molecular tools to detect <i>Aegilops</i> 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 <i>in situ</i> hybridization (FISH) maps for M- and U-genome progenitors, <i>Aegilops comosa</i> and <i>Aegilops umbellulata</i>, respectively. Forty-three ortholog genes were located on 47 loci in <i>Ae. comosa</i> and on 52 loci in <i>Ae. umbellulata</i> 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 <i>Ae. umbellulata</i> 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 <i>Aegilops markgrafii</i>. To develop molecular markers with exact physical positions on chromosomes of <i>Aegilops</i>, the single-gene FISH data were validated <i>in silico</i> 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 <i>Ae. comosa</i> and 40 of the 52 map positions in <i>Ae. umbellulata</i>. Polymorphic regions, thus, identified enabled the development of molecular markers, which were PCR validated using wheat-<i>Aegilops</i> disomic chromosome addition lines. The single-gene FISH-based approach allowed the development of PCR markers specific for cytogenetically mapped positions on <i>Aegilops</i> chromosomes, substituting as yet unavailable segregating map. The new knowledge and resources will support the efforts for the introgression of <i>Aegilops</i> genes into wheat and their cloning.