We applied fluorescent in situ hybridization (FISH) using (GAA)10 microsatellite repeat alone or in combination with oligo pSc119.2-1, pTa535-1, pAs1-1, 5S and 45S rDNA probes to identify individual chromosomes and produce banded karyotypes for five allopolyploid Aegilops species that were thought to possess M subgenome. Labelling patterns of GAA repeats allow identification of individual chromosomes in the studied species including diploid Ae. comosa subsp. heldreichii (2n = MhMh) and Ae. umbellulata (2n = UU); and allopolyploid Ae. biuncialis (2n = UbUbMbMb), Ae. geniculata (2n = UgUgMgMg), Ae. columnaris (2n = UcUcXcXc), Ae. neglecta (2n = UnUnXnXn) and Ae. crassa (2n = 4x = D1D1XcrXcr and 2n = 6x = D1D1XcrXcrDcrDcr). Intraspecific polymorphisms of GAA labelling patterns were observed between different genotypes in each species. FISH results showed that the X* subgenomes of Ae. columnaris and Ae. neglecta are highly similar, implying that both species originated from a common ancestor. However, these X* subgenomes were also distinct from and more heterochromatic when compared to the M* subgenomes of Ae. biuncialis and Ae. geniculata. This suggests that the X* subgenomes may not have originated from the Ae. comosa, as previously thought. Although (GAA)n bands are relatively few and weak in Ae. tauschii (DD genome), all 4x Ae. crassa (D1D1XcrXcr genome) chromosomes showed distinct GAA patterns. (GAA)10 probe alone was not sufficient for the discrimination of all the chromosomes of 6x Ae. crassa (D1D1XcrXcrDcrDcr), which needs a combination of (GAA)10 with the pTa535-1 or pAs1-1 probes.
