**5. Conclusions**

The mini-satellite sequences are abundantly distributed in the wheat genome, which served as powerful cytogenetic and genomic markers for germplasm enhancement and breeding practices. The present study identified a novel mini-satellite repeat Ta-3A1, and revealed its copy number and physical locations of Ta-3A1 among chromosomes of wheat and their ancestral genomes. The large sequence variation of Ta-3A1 displayed rapid evolution of mini-satellites occurred during speciation and polyploidization The Ta-3A1 based ND-FISH may be helpful for chromosome identification of wheat and related species, which can be directly used to wheat improvement by chromosome engineering.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4395/9/2/60/s1, Table S1: List of materials and their origins; Figure S1: The interpretation of TRs by numbers of individual copy, array, and cluster; Figure S2: Phylogenetic tree and sequence comparison of the Ta-3A1 repeats.

**Author Contributions:** Z.Y. (Zujun Yang) and G.L. designed the experiments. T.L., G.L., Z.Y. (Zhihui Yu), J.M. and Q.C. performed the experiments. Z.Y. (Zujun Yang), E.Y., and T.L. analyzed the data. Z.Y. (Zujun Yang) and G.L. wrote the paper.

**Funding:** This research was funded by the National Key Research and Development Program of China (2016YFD0102000), the Applied and Basic Project (2016JY0075) from the Science and Technology Department of Sichuan, China, and the National Natural Science Foundation of China (No. 31171542).

**Acknowledgments:** We are thankful to Ian Dundas at the University of Adelaide, Australia, for the helpful comments on the manuscript.

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