**5. Conclusions**

In conclusion, our results showed that the soybean genome contains 61 *XTH* genes, the largest family of XTH proteins characterized in any organism to date. The results of phylogenetic analysis and chromosome location/structure provide an overview of the soybean *GmXTH* gene family. The results of the segmental and tandem duplication during expansion of the *GmXTH* gene family provide a genome-wide evolutionary overview. The results of conserved amino acid motif analysis and expression pattern analysis further provide insight into their putative function. Additionally, functional analysis of *AtXTH31* in a heterologous system suggests that the higher germination rate and longer roots/hypocotyls induced by the increased XTH activity may be responsible for the flooding tolerance of transgenic plants. Further comprehensive experiments may be required to elucidate the cellular locations and functions to understand the biological role of XTHs in soybean.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/ 9/2705/s1. Supplementary Table S1: Information related to the 61 genes homologous to XTH genes in the soybean genome; Supplementary Table S2: Oligonucleotide primer sequences used in this work; Supplementary Table S3: Estimate of the dates of the segmental duplication events of the soybean XTH gene pairs; Supplementary Figure S1: Exon-intron structure of soybean *XTHs*; Supplementary Table S4: Search for cis-elements in the GmXTH gene promoters.

**Author Contributions:** L.S. designed the experiments, analyzed the data, and prepared the manuscript. B.V. worked on the gene clone and vector construction. S.P. worked on the transgenic soybean phenotype analysis. J.W. analyzed the digital PCR data. H.T.N. conceived and supervised the project. All authors have read, revised, and approved the manuscript.

**Acknowledgments:** We acknowledge Missouri Soybean Merchandising Council, USA and Yang Zhou Science and Technology Bureau (YZ2018156), China for financial support and Plant Transformation Core Facility at the University of Missouri for soybean transformation. We thank Thermo Scientific (Life Technology) for supporting the Q3D PCR equipment. We thank Caifu Chen, Yalei Wu, Pius Brzoska, and David Keys for digital PCR technical support. We thank Yongqin Wang and Chenglin Chai for helping with plant hormone treatment. We thank Mackensie Murphy and Raymond Mutava for their help on the transgenic plant flooding assay.

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

### **References**


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