| Summary: | <i>Aegilops tauschii</i> Coss. (2n = 2x = 14, DD) is a problematic weed and a rich source of genetic material for wheat crop improvement programs. We used physiological traits (plant height, dry weight biomass, Na<sup>+</sup> and K<sup>+</sup> concentration) and 14 microsatellite markers to evaluate the genetic diversity and salinity tolerance in 40 <i>Ae. tauschii</i> populations. The molecular marker allied with salinity stress showed polymorphisms, and a cluster analysis divided the populations into different groups, which indicated diversity among populations. Results showed that the expression level of <i>AeHKT1;4</i> and <i>AeNHX1</i> were significantly induced during salinity stress treatments (50 and 200 mM), while <i>AeHKT1;4</i> showed relative expression in roots, and <i>AeNHX1</i> was expressed in leaves under the control conditions. Compared with the control conditions, the expression level of <i>AeHKT1;4</i> significantly increased 1.7-fold under 50 mM salinity stress and 4.7-fold under 200 mM salinity stress in the roots of <i>Ae. tauschii</i>. <i>AeNHX1</i> showed a relative expression level of 1.6-fold under 50 mM salinity stress and 4.6-fold under 200 mM salinity stress compared with the control conditions. The results provide strong evidence that, under salinity stress conditions, <i>AeHKT1;4</i> and <i>AeNHX1</i> synergistically regulate the Na<sup>+</sup> homeostasis through regulating Na<sup>+</sup> transport in <i>Ae. tauschii</i>. <i>AeNHX1</i> sequestrated the Na<sup>+</sup> into vacuoles, which control the regulation of Na<sup>+</sup> transport from roots to leaves under salinity stress conditions in <i>Ae. tauschii</i>.
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