**4. Discussion**

Knowledge regarding the influence of the amylose/amylopectin ratio on starch properties has encouraged the search for allelic variants that could increase/decrease either starch component. The most studied, in this context, has been the ADP glucose starch glycosyl transferase (GBSSI or the waxy proteins) solely responsible for amylose synthesis. This starch synthase has been studied in several cereal species, mainly those in which the starch properties are important for their use in the agri-food industry or in bio-ethanol production [46].

Recently, biotechnological techniques have allowed the development of new species using phylogenetically related species. These new species could also be used as a bridge to transfer new variations to common wheat. Thus, *H. chilense*, as a species involved in the synthesis of tritordeum, could be useful [25]. The incorporation of the *Hc<sup>h</sup>* genome in durum wheat has clear effects on the quality characteristics of the tritordeum. For example, the presence of the glutenins or hordeins of this wild species modifies the strength of the gluten in tritordeum flour [22], and their hordoindolines

change the texture of the grain from the ultra-hard of the parent durum wheat to soft in the derived tritordeum [29].

Here, we have studied one of the main keys in cereal flour quality, starch, by molecularly characterising the *Wx* gene in the two main lines of *H. chilense* used in the development of the tritordeums [25]. The *H. chilense* waxy proteins presented structures very similar to those of waxy proteins in other species of *Triticeae*, such as wheat and barley. The sizes of the predicted proteins were similar, although numerous amino acid changes were detected. However, these changes were mostly silent and not related to the active site of this enzyme, and probably, without influence on its function. In fact, some of these changes have been observed in other *Wx* genes [13]. The highly conserved structure of this gene makes it a good candidate for phylogenetic analysis [11,45,47–51]. In this study, the use of *Ka* established the separation between the *Hordeum* genomes at ~3 MYA.

In barley, Kramer and Blander [52] located the *Wx* gene on the short arm of chromosome 1 (7H). In common wheat, the waxy loci are located on chromosome 7AS (*Wx*-A1), chromosome 4AL, which was translocated from the original 7BS, (*Wx*-B1) and chromosome 7DS (*Wx*-D1) [10]. Here, the *Wx-Hch*1 gene from *H. chilense* was located on 7 *HchL*, opposite the arm location found in the other Triticeae species [13]. Mattera et al. [53] indicated a similar change in the location of the *Phytoene syntase* (*Psy-1*) gene in *H. chilense. Psy*-1 was mapped in the distal region of 7HchS, while this gene was located on the opposite arm of chromosome 7 in other Poaceae species [14]. On the basis of these changes, Mattera et al. [53] suggested that an inversion occurred between the distal parts of 7 *HchS* and 7 *HchL*, which has been confirmed by Avila et al. [54]. The location of the *Wx*-*Hch*1 gene on 7 *HchL* in the present study supports this hypothesis on a structural change involving the distal regions of *H. chilense* chromosome 7 *Hch*.
