**4. Discussion and Conclusions**

Genomes of modern wheat cultivars and landraces are shaped by inter- and intra-species introgression events [31]. The functional consequences of the introgressions and their e ffect on phenotypic variation and traits beneficial to humans, such as bread-making quality, have been poorly explored. Our study showed that *wbm* is a part of genomic introgression of unknown scale into the long arm of chromosome 7A. The protein and nucleotide sequences of *wbm* are phylogenetically distant from the homologous sequence of the A genome donor species, pointing to its distinct origin from unknown species. Concluding whether the introgression was a result of interspecies hybridization during the wheat breeding process or if it existed before the origin of wheat is di fficult. Furtado et al. [11] reported the presence of *wbm* only in some genotypes of *T. monococcum* and *T. urartu*, suggesting that both scenarios are possible. With such a high divergence rate of *wbm* locus, the resequencing and read mapping to the reference genome (CS cultivar) will not allow identifying the origin of *wbm,* as it requires substantial similarity between reference and target genomes. Therefore, ongoing de novo sequencing and genome assembly of wheat cultivars (10+ Genome Project, http://www.10wheatgenomes.com/) and closely-related species are essential to identify the introgression similar to *wbm*.

The function of *wbm* protein remains elusive. Based on the specific spatiotemporal expression pattern and cysteine content of *wbm* protein, Furtado et al. [11] hypothesized that *wbm* protein can influence breadmaking quality through the generation of inter and intramolecular disulfide bonds. Notably, *wbm* protein is encoded by a small open reading frame (sORF). sORFs underwent rapid genomic evolution and may occur de novo in the genomes [32]. Comparison of the *wbm* protein sequence demonstrated high divergence between closely related species, consistent with general patterns of sORF evolution. The length of putative *wbm*-like proteins from the CS cultivar and closely related species also di ffer from the *wbm* protein. Although the molecular mechanism of *wbm* function is unknown, whether the sequence divergence is coupled with changes in *wbm* function is a topic that requires future research.

The established location of *wbm* on the A genome enabled the screening of a collection of triticale genotypes possessing A, B, and R genomes, and we found only three lines possessing *wbm*. Although a number of desirable traits in triticale have been inherited from both parents [13,14], the genes controlling desirable dough properties are located on the D genome and therefore are not present in hexaploid triticale [14]. Hence, new genetic resources are required to produce triticale producing strong gluten with high extensibility. Storage proteins of triticale, secaloglutenins, include a combination of HMW and LMW glutenins from the A and B genomes, secalins from the R genome, and α-, β-, and γ-gliadins [33,34]. Triticale lines possessing *wbm* may have unique gluten properties if *wbm* is capable of generating links between di fferent storage proteins. However, further testing of this hypothesis requires a substantial amount of seeds to perform the baking test for these lines, which is the goal for our future research.

Here, we showed that GWseqVar3, previously described as an allelic variant of *wbm* [11], is orthologous to *wbm-like* located on chromosome 7A of wheat. In turn, *wbm-like* the CS cultivar is most probably a pseudogene surrounded by insertion of mobile elements, which has a negligible expression in grain. We found that *wbm* previously introgressed into the genomes of some wheat and triticale cultivars from unknown species. Elucidating the evolutionary relationships between *wbm* and its homologous sequences in wheat and closely related species allowed us to demonstrate that *wbm* protein is highly diverged, except its N-terminal. Finally, the location of *wbm* in the A genome

offered the opportunity to select three triticale lines carrying this gene that can be further exploited in the future for improving the bread-making quality of this crop.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4395/9/12/854/s1, Table S1: the list of triticale genotypes used in this study.

**Author Contributions:** Conceptualization, I.K. and A.S.; methodology, I.K.; formal analysis, A.P., N.M., M.D., G.K., M.K., I.G., and S.S.; writing—original draft preparation, I.K.; writing—review and editing, I.K., L.K., and A.S.; funding acquisition, A.S., G.K.

**Funding:** This research was funded by the Ministry of Education and Science of Russian Federation (goszadanie № 0431-2019-0005).

**Conflicts of Interest:** The authors declare no conflicts of interest.
