*2.4. Comparative Mapping*

The orthologous relationship between the 2A, 2B, and 2D genome chromosomes of bread wheat and the 2Hc<sup>h</sup> chromosome from *H. chilense* has been studied from the genomic perspective of wheat as described previously [30]. For the construction of the physical map, the expressed sequence tag (EST) source sequences (File S2) were used as queries in BLASTn searches against the wheat reference pseudomolecules [31] to identify the start positions (bp) of the ESTs. In this study, BLAST hits with *E* values smaller than 1e−10, identity % > 58.44 and alignment length > 100 bp were considered significant. The genomic start positions in bp of the best hits in wheat pseudomolecules (File S3) were used to construct a physical map of the polymorphic COS markers. The wheat reference genome sequence [31] was used to determine the centromere positions for 2A, 2B and 2D wheat chromosomes. Both the length in bp of wheat pseudomolecules, as well as the start genomic positions of the ESTs, were converted to pixels. Then, the data from the BLASTn searches were used to construct a physical map for 2A, 2B, and 2D wheat chromosomes showing the position of the source EST of the COS markers assigned to *H. chilense* chromosome 2Hch.

The rice locus (RAP) [21] was used to locate the COS markers in the barley genome zipper [32]. The RAP locus identifier was retrieved using the ID Converter tool [33]. The full-length barley cDNA corresponding to each rice locus was used for determination of the barley Unigene corresponding to each COS marker. The Unigene sequences were aligned in Barleymap [34] to obtain their positions in the International Barley Sequencing Consortium map [32,35].
