Allelic variants of OsHKT1;1 underlie the divergence between indica and japonica subspecies of rice (Oryza sativa) for root sodium content

Salinity is a major factor limiting crop productivity. Rice (Oryza sativa), a staple crop for the majority of the world, is highly sensitive to salinity stress. To discover novel sources of genetic variation for salt tolerance-related traits in rice, we screened 390 diverse accessions under 14 days of moderate (9 dS·m^-1) salinity. In this study, shoot growth responses to moderate levels of salinity were independent of tissue Na⁺ content. A significant difference in root Na⁺ content was observed between the major subpopulations of rice, with indica accessions displaying higher root Na⁺ and japonica accessions exhibiting lower root Na⁺ content. The genetic basis of the observed variation in phenotypes was elucidated through genome-wide association (GWA). The strongest associations were identified for root Na⁺:K⁺ ratio and root Na⁺ content in a region spanning ~575 Kb on chromosome 4, named Root Na⁺ Content 4 (RNC4). Two Na⁺ transporters, HKT1;1 and HKT1;4 were identified as candidates for RNC4. Reduced expression of both HKT1;1 and HKT1;4 through RNA interference indicated that HKT1;1 regulates shoot and root Na⁺ content, and is likely the causal gene underlying RNC4. Three non-synonymous mutations within HKT1;1 were present at higher frequency in the indica subpopulation. When expressed in Xenopus oocytes the indica-predominant isoform exhibited higher inward (negative) currents and a less negative voltage threshold of inward rectifying current activation compared to the japonica-predominant isoform. The introduction of a 4.5kb fragment containing the HKT1;1 promoter and CDS from an indica variety into a japonica background, resulted in a phenotype similar to the indica subpopulation, with higher root Na⁺ and Na⁺:K⁺. This study provides evidence that HKT1;1 regulates root Na⁺ content, and underlies the divergence in root Na⁺ content between the two major subspecies in rice.