*2.1. Plant Materials*

Seeds of two *H. chilense* lines (H1 and H7) that were self-pollinated for two generations were used in this study. The ditelosomic addition lines (CS + 7*HchS* and CS + *7HchL*), together with both parental lines (common wheat cv. "Chinese Spring" and line H1) were used to locate the *Wx* gene in *H. chilense*. These materials were grown in greenhouse conditions.

### *2.2. DNA Extraction and PCR Amplification*

For DNA extractions, ~100 mg of young leaf tissue was excised and immediately frozen in liquid nitrogen. DNA was isolated using the cetyltrimethyl ammonium bromide (CTAB) method as described by Stacey and Isaac [32].

The primers BDFL (5--CTGGCCTGCTACCTCAAGAGCAACT-3-) and BRD (5--CTGACGTCCATGCCGTTGACGA-3-) designed by Nakamura et al. [33] were used to detect the presence of the *Wx-Hch1* gene in the ditelosomic addition lines. The amplification was performed in a 20 μL final reaction volume, containing 50 ng of genomic DNA, 1.25 mM MgCl2, 0.2 mM dNTPs, 4 μL 10× PCR bu ffer, 0.2 μM of each primer and 0.75 U GoTaq ® G2 Flexi DNA polymerase (Promega). The PCR conditions included an initial denaturation step of 3 min at 94 ◦C followed by 35 cycles as follows: 30 s at 94 ◦C, 30 s at 65 ◦C then 2 min at 72 ◦C. After the 35 cycles, a final extension of 5 min at 72 ◦C was included.

Amplification products were fractionated in vertical PAGE gels with 8% polyacrylamide concentration ( *<sup>w</sup>*/*<sup>v</sup>*, C: 1.28%) and the bands were stained with GelRed ™ nucleic acid staining (Biotium) and visualized under UV light.

### *2.3. Cloning of PCR Products and Sequencing Analysis*

Owing to the length and structure of the *Wx* gene, ~2800 bp with 11 introns and 12 exons, three fragments were amplified using primers designed by Guzmán and Alvarez [34]. The first fragment includes the first to third exons (Wx1Fw: 5--TTGCTGCAGGTAGCCACACC-3- and Wx1Rv: 5--CCGCGCTTGTAGCAGTGGAA-3-), the second extends from the third to the sixth exon (Wx2Fw: 5--ATGGTCATCTCCCCGCGCTA-3- and Wx2Rv: 5--GTTGACGGCGAGGAACTTGT-3-), and the last fragment covers the region spanning the 6th to the 11th exon (Wx3Fw: 5--GGCATCGTCAACGGCATGGA-3- and Wx3Rv: 5--TTCTCTCTTCAGGGAGCGGC-3-).

All amplifications were performed in 50 μL final volumes, containing 100 ng of DNA genomic, 1.25 mM MgCl2, 0.2 mM dNTPs, 10 μL 10× PCR buffer and 0.75 U GoTaq®G2 Flexi DNA polymerase (Promega). The primer concentrations were 0.4, 0.3 and 0.2 μM per primer for the first, second and third fragments, respectively. The PCR conditions included an initial denaturation step of 3 min at 94 ◦C and then 35 cycles as follows: for Wx1Fw/Wx1Rv, 40 s at 94 ◦C, 30 s at 64 ◦C and 1 min at 72 ◦C, for Wx2Fw/Wx2Rv, 30 s at 94 ◦C, 30 s at 66 ◦C and 90 s at 72 ◦C, and for *Wx*3*Fw*/*Wx*3*Rv*, 40 s at 94 ◦C, 30 s at 62 ◦C and 90s at 72 ◦C. After the 35 cycles, all reactions included a final extension of 5 min at 72 ◦C.

The PCR products were purified by separation in 1% agarose gel, excised and then independently ligated into the pSpark®-TA Done vector (Canvax). They were then transformed into *Escherichia coli* 'CVX5α' competent cells (Canvax). Inserts were sequenced by Sanger method from at least three different clones. The novel sequences are available from the GenBank database [*Wx-Hch1a*: MK045501 for the H1 line, and *Wx-Hch1b*: MK045502 for the H7 line].
