**3. Results**

### *3.1. Cytogenetic and Molecular Characterization of Wheat—H. chilense Introgression Lines Involving Chromosome 2Hc<sup>h</sup>*

The pAs1 and *H. chilense* genomic DNA used as probes in FISH analysis allowed the identification of a pair of 2Hc<sup>h</sup> chromosomes and the absence of the wheat 2D chromosome pair in one line with 42 chromosomes. This result indicated that this line was disomic for the substitution 2Hch(2D) (Figure 1a). The absence of 2D was tested using *Xgwm261-2DS* (Figure 2a) and *Xgwm157-2DL* (Figure 2b) molecular markers. A pair of telocentric chromosomes was identified by FISH in one line with 42 + 2t chromosomes (Figure 1b). To determine the chromosome arm involved in each introgression line, we used the *c749557* COS marker mapped on the 2HchS arm and *c731690* mapped on 2HchL, respectively (see Section 3.2). The presence of the c731690 marker for 2HchL and the absence of the *c749557* marker for 2HchS showed that this line was ditelosomic for the 2HchL arm (Figure 3a,b).

FISH analysis revealed a line apparently carrying chromosome 2Hc<sup>h</sup> (Figure 1c). Marker *c731690* for 2HchL was amplified in this line, but there was no amplification of the *c749557* marker for 2HchS (Figure 3a,b). These results suggested that a 2HchL·2HchL isochromosome was present in this line, and it was named Iso 2HchL. Both the ditelosomic 2HchL and Iso 2HchL lines were nullisomic for chromosome 2D, as demonstrated by the absence of amplification of both *Xgwm261-2DS* (Figure 2a) and *Xgwm157-2DL* (Figure 2b) molecular markers.

We identified two lines carrying centromeric translocations involving the 2HchS chromosome arm and wheat chromosomes. One of these lines was homozygous for the 2HchS·2DL translocation (Figure 1d). The other translocation line was a double monosomic for 2HchS·2DL and 7HchS·D translocations (Figure 1e). Chromosome-specific SSR markers confirmed the absence of 2DS (Figure 2a) and the presence of 2DL (Figure 2b) in both lines. Amplification of the *c749557* marker (Figure 3a) and the absence of amplification of the *c731690* marker (Figure 3b) demonstrated the presence of 2HchS and the absence of 2HchL, respectively, in both lines. COS markers *c779791* and *c759439*, previously assigned to 7HchS and 7HchL, respectively [18], were used to detect introgression from chromosome 7Hc<sup>h</sup> (Figure 3c,d). Amplification of the *c779791* marker specific for the 7HchS arm (Figure 3c) indicated the presence of 7HchS translocated to an unidentified wheat fragment. The presence of pAs1 signals on the wheat small fragment indicated that the chromosome 7HchS arm was translocated to an unidentified D-genome chromosome (Figure 1e). The absence of amplification of chromosome-specific markers *Xcfd66*-7DS and *Xbarc111*-7DL demonstrated the absence of a 7D chromosome pair in this line (Figure 2c,d).

In the remaining two lines, two centromeric translocations involving 2Hc<sup>h</sup> and 7Hc<sup>h</sup> *H. chilense* chromosomes were detected. One line was homozygous for the 7HchS·2HchL translocation (Figure 1f) and the other one was monosomic for the 2HchL·7HchL translocation. Both translocation lines were nullisomic for chromosome 2D (Figure 2a,b). Chromosome-specific SSR marker patterns for 2D (*Xgwm261-2DS* and *Xgwm157-2DL*) and 7D (*Xcfd66-7DS* and *Xbarc111-7DL*) genome chromosomes are given in Figure 2a,b and Figure 2c,d, respectively. Chromosome-specific marker results for chromosome 7HchS and 7HchL are given in Figure 3c,d. Table 1 shows the chromosome constitutions of all the *H. chilense* introgression lines. All lines were vigorous and seed set.

**Figure 1.** Fluorescence in situ hybridization (FISH) with the pAs1 repetitive (red) and *H. chilense* genomic DNA (green) probes to mitotic metaphase of wheat—*H. chilense* introgression lines involving chromosome 2Hch. (**a**) Disomic substitution 2Hc<sup>h</sup> (2D); (**b**) Ditelosomic 2HchL; (**c**) Isochromosome 2HchL; (**d**) Translocation 2HchS·2DL; (**e**) Translocation 2HchS·2DL + T7HchS·D; (**f**) Translocation 7HchS·2HchL; (**g**) Translocation 2HchL·7HchL. Bar = 10 μm.

**Figure 2.** Molecular characterization of introgression lines with wheat chromosome-specific simple sequence repeats (SSR)markers. (**a**)*Xgwm261*-2DS; (**b**)*Xgwm157*-2DL; (**c**)*Xcfd66*-7DS and (**d**)*Xbarc111*-7DL.

**Figure 3.** Examples of PCR amplification profiles used for identifying chromosome 2Hc<sup>h</sup> and 7Hc<sup>h</sup> arms in the introgression lines. (**a**) c749557 mapped on the short arm of chromosome 2Hch; (**b**) c731690 mapped on the long arm of chromosome 2Hch; (**c**) c779791 mapped on the short arm of chromosome 7Hch; and (**d**) c759439 mapped on the long arm of chromosome 7Hch. "Chinese Spring" (CS), *H. chilense* (Hch), ditelo 2HchS, ditelo 7HchS, ditelo 7HchL and disomic substitution line CS 7Hch(7D) were used as controls.


**Table 1.** Chromosome constitutions of wheat—*H. chilense* introgression lines involving chromosome 2Hch.

1 disomic;2 monosomic; 3 doublemonosomic

### *3.2. Transferability and Chromosome Location of COS Markers in H. chilense*

The transferability to *H. chilense* of 83 COS markers from wheat homoeologous group 2 was studied (File S1). First, all 83 markers were screened for polymorphisms (size polymorphisms or presence and absence) between *H. chilense* and common wheat. Of the 83 markers, 65 (78.3%) consistently amplified *H. chilense* products and 26 (40.0% of the total) were polymorphic between *H. chilense* and wheat (Table 2). Twenty-four of these 26 polymorphic markers were mapped to chromosome 2Hch, as demonstrated by their presence in the wheat—*H. chilense* 2Hch(2D) substitution line. We were unable to map the remaining two markers because they did not amplify products in any of the available wheat—*H. chilense* addition lines. Of the 24 COS markers mapped on chromosome 2Hch, eight were located on 2HchS and 16 were located on 2HchL, as demonstrated by their presence and absence in 2HchS or 2HchL ditelosomic lines, respectively. Table 2 summarize the characterization and chromosome arm location of wheat COS markers on *H. chilense* chromosome 2Hch. Figure 3a,b shows examples of amplification of homoeologous group 2 COS markers.

**Table 2.** Characterization and chromosome localization of wheat conserved orthologous set (COS) markers on *H. chilense* chromosome 2Hch.



**Table 2.** *Cont*.

1 Quraishi et al. (2009); 2 Position in barley determined using Barleymap (http://floresta.eead.csic.es).

### *3.3. Wheat—H. chilense Group 2 Homoeology*

To investigate wheat—*H. chilense* group 2 macrosyntenic relationships, the source ESTs of the 24 polymorphic COS markers mapped on chromosome 2Hc<sup>h</sup> were BLASTed to the sequences of the wheat chromosomes [31]. All EST markers showed hits on wheat pseudomolecules (File S3). To produce a physical map (Figure 4), the start positions of the alignments of the best hits on the A, B, and D genomes were extracted. All markers assigned to 2HchS and 2HchL were located in the same arm of wheat homoeologous group 2 chromosomes (Table 2, Figure 4).

To determine the positions of COS markers in Barley-maps (Table 2) the align tool implemented in Barleymap [34] was used to determine the positions of COS markers in barley maps (Table 2). Four of the COS markers were not found in the barley genome zipper [32] so their position in barley could not be determined. A good correspondence between *H. chilense* and barley was found for the arm locations of COS markers (Table 2).

**Figure 4.** Visualization of wheat-*H. chilense* orthologous regions from the perspective of wheat homoeologous chromosome group 2. Physical map of the source expressed sequence tags (ESTs) of the COS-markers (left), the genomic positions on wheat pseudomolecules (kb) are on the right.
