*2.3. Molecular Marker Analysis*

A total of 82 COS markers from wheat homoeologous group 2 [21] were studied for their utility in *H. chilense* (File S1). *H. chilense* (line H7) and common wheat CS were used as controls. The CTAB method [26] was used for DNA extraction of young leaf tissue. The concentration of each sample was estimated using a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Amplifications were made using a TGradient thermocycler (Biometra, Göttingen, Germany) with 60 ng of template DNA in a 25 μl volume reaction containing 5 μl of 10× PCR Buffer, 0.5 μM of each primer, 1.5–2.0 mM MgCl2, 0.3 mM dNTPs and 0.25 U of Taq DNA polymerase (BIOTOOLS B&M Laboratories, Madrid, Spain). The PCR conditions of COS markers were as follows: 4 min at 94◦C, followed by 35 cycles of 45 s at 94◦C, 50s at 58◦C annealing temperature, 50 s at 72◦C, and a final extension step of 7 min at 72 ◦C.

In addition, four chromosome-specific SSR markers for the wheat D-genome were used for molecular characterization of the introgression lines [27,28]. *Xgwm261* and *Xgwm157* markers were used to detect 2DS and 2DL chromosome arms, respectively. *Xcfd66* and *Xbarc111* were used to detect 7DS and 7DL chromosome arms, respectively. Amplifications were carried out as described at GrainGenes [29] One plant from each introgression line was used for the molecular characterization. "Chinese Spring", *H. chilense*, a ditelosomic 2HchS line, a ditelosomic 7HchS line, a ditelosomic 7HchL line and disomic substitution line CS 7Hch(7D) were used as controls. Ditelosomic 2HchS and ditelosomic 7HchS lines were provided by the John Innes Centre (UK). The ditelosomic 7HchL line and disomic CS 7Hch(7D) substitution line were obtained previously [17].

The amplified products were resolved using 2% agarose gels (SSRs) or polyacrylamide gels (10%, w/v; C: 2.67%) (COS) and stained with ethidium bromide or SafeView Nucleic Acid Stain (NBS Biologicals, Huntingdon, UK) incorporated in the gel. A 100 bp DNA ladder (Solis BioDyne, Tartu, Estonia) was used as a standard molecular weight marker. Kodak Digital Science 1D software (version 2.0) was used to determine the amplicon lengths.
