**5. Conclusions**

With the application of GBS, it has been possible to generate 45,507 SNP markers for a diversity analysis of crested wheatgrass. The variation residing among these 12 lines of CWG was found to be 15.8%. Further analysis grouped the assayed samples into four genetic clusters, and revealed the genetic distinctness of two cultivars each from Kazakhstan and Russia, respectively. These results can enhance parental selection for increased genetic variation and improved offspring performance in crested wheatgrass breeding. The findings in this study can also aid in the application of GBS in the characterization of non-model plants with complex genomes.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/9/ 2587/s1.

**Author Contributions:** Y.-B.F., B.B. and B.C. conceived the project; Y.-B.F. designed research; B.C. prepared the study material; Y-B.F. conducted sequencing; K.B. and Y.-B.F. performed data analysis; K.B. wrote the manuscript; B.C., Y.-B.F. and B.B. made revisions to the manuscript. All authors read and approved the final manuscript.

**Funding:** The work was financially supported by the Beef Cattle Research Council of Canada and Agriculture and Agri-Food Canada (AAFC) Growing Forward 2 Funds (FGR.08.13).

**Acknowledgments:** The author would like to thank Gregory Peterson and Carolee Horbach for their technical assistance; Isobel Parkin for the access to and the use of the Illumina MiSeq instrument; Compute Canada and Westgrid for providing the high-performance computing service as well as of their technical support; and Helen Booker, Bunyamin Tar'an and two anonymous journal reviewers for their helpful comments on the early version of the manuscript.

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