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Background  The annual allotetraploid species  Aegilops geniculata  harbors a number of traits relevant for wheat improvement. An effective cytogenetic method has yet to be developed to distinguish between each of its 14 chromosomes.    Results  A fluorescence in situ hybridization (FISH) approach was adopted to describe the karyotype of  Ae. geniculata.  Each of its 14 chromosomes was unequivocally recognized using a cocktail of three probes, namely pTa-713, (AAC) 5  and pTa71. FISH karyotyping was then used to detect and characterize selections from an  Ae. geniculata  × bread wheat wide cross of a chromosome 1M g  disomic addition line and three 4M g (4B) substitution lines. The identity of the addition line was confirmed by the presence of  Glu-M1 , detected both using an SDS-PAGE separation of endosperm proteins and by applying a PCR assay directed at the  Glu-M1  locus. The status of the substitution lines was validated by genotyping using a wheat single nucleotide polymorphism chip.    Conclusion  FISH karyotyping based on pTa-713, (AAC) 5  and pTa71 will be useful for determining the contribution of  Ae. geniculata  to derivatives of an  Ae. geniculata  × wheat wide cross. SNP chip-based genotyping is effective for confirming the status of whole chromosome wheat/alien substitution lines.    Electronic supplementary material  The online version of this article (10.1186/s13039-019-0428-2) contains supplementary material, which is available to authorized users.

The karyotype of Aegilops geniculata and its use to identify both addition and substitution lines of wheat