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

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 and characterization of Triticum turgidum–Aegilops umbellulata amphidiploids

The U genome of Aegilops umbellulata is an important basic genome of genus Aegilops. Direct gene transfer from Ae. umbellulata into wheat is feasible but not easy. Triticum turgidum–Ae. umbellulata amphidiploids can act as bridges to circumvent obstacles involving direct gene transfer. Seven T. turgidum–Ae. umbellulata amphidiploids were produced via unreduced gametes for spontaneous doubling of chromosomes of triploid T. turgidum–Ae. umbellulata F1 hybrid plants. Seven pairs of U chromosomes of Ae. umbellulata were distinguished by fluorescence in situ hybridization (FISH) probes pSc119.2/(AAC)5 and pTa71. Polymorphic FISH signals were detected in three (1U, 6U and 7U) of seven U chromosomes of four Ae. umbellulata accessions. The chromosomes of the tetraploid wheat parents could be differentiated by probes pSc119.2 and pTa535, and identical FISH signals were observed among the three accessions. All the parental chromosomes of the amphidiploids could be precisely identified by probe combinations pSc119.2/pTa535 and pTa71/(AAC)5. The T. turgidum–Ae. umbellulata amphidiploids possess valuable traits for wheat improvement, such as strong tillering ability, stripe rust resistance and seed size-related traits. These materials can be used as media in gene transfers from Ae. umbellulata into wheat.