*2.1. Plant Material*

A "Chinese Spring" (CS) wheat—*H. chilense* double 2Hch(2D)-7Hch(7D) disomic substitution line, previously obtained at the University of Córdoba (results not shown), was used for inducing structural changes in chromosome 2Hc<sup>h</sup> using gametocidal chromosome 2Cc from *Aegilops cylindrica* host. The double 2Hch(2D) and 7Hch(7D) disomic substitution line was obtained by pollinating tritordeum (the fertile amphiploid between *H. chilense* and *T. turgidum* L., AABBHchHch, 2*n* = 6x = 42) with a wheat disomic addition line for gametocidal chromosome 2Cc from *Ae. cylindrica* Host. following the breeding procedure described in [8]. The double substitution 2Hch(2D) and 7Hch(7D) line was pollinated with the wheat disomic addition line for the gametocidal chromosome 2Cc from *Ae. cylindrica*. The F1 plants monosomic for 2Hch, 7Hc<sup>h</sup> and 2Cc were selfed for four generations.

### *2.2. Fluorescence In Situ Hybridization (FISH)*

The excised root tips were pretreated with ice water for 24 h and then fixed in acetic ethanol: acetic acid (3:1, v/v), as described previously [8]. The FISH protocol was carried out as described by [22]. The pAs1 sequence (1 kb) isolated from *Aegilops tauschii* Coss. [23] and *H. chilense* genomic DNA were used as probes. The pAs1 probe hybridizes to D-genome chromosomes of wheat [24] and Hch-genome chromosomes from *H. chilense* [25]. The pAs1 probe and *H. chilense* DNA were labeled with biotin-16-dUTP (Roche Diagnostics, Switzerland) and with digoxigenin-11-dUTP (Roche Diagnostics, Switzerland), respectively, by nick translation. Three plants per each introgression line were analyzed.

Biotin- and digoxigenin-labelled probes were detected with streptavidin-Cy3 conjugates (Sigma, St. Louis, MO, USA) and antidigoxigenin FITC (Roche Diagnostics) antibodies, respectively. The

chromosomes were counterstained with DAPI (4-,6-diamidino-2-phenylindole) and mounted in Vectashield mounting medium (Vector laboratories, Inc., Burlingame, CA, USA). A Leica DMR epifluorescence microscope was used for signal visualization. Images were captured with a Leica DFC7000T camera and processed with LEICA application suite v4.0 software (Leica, Germany).
