Dissection of Seven Saline Tolerance Genes in a Geng/Japonica Population Suggests the Subspecies-Specific Differentiation at RST1530A in Rice

Improving salt tolerance (ST) is a critical objective for rice production in China’s Huang–Huai–Hai (3H) region, where soil salinity poses a persistent threat to crop productivity. Natural allelic variations in ST genes serve as vital genetic resources for addressing this challenge. In this study, we investigated nine single nucleotide polymorphisms (SNPs) associated with seven known ST genes across 215 geng/japonica rice varieties from this region, including OsSTG5, SKC1, OsHKT1;1, OsHKT2;3, OsWRKY53, OsSTL1, and RST1. These genes cover diverse functional categories—ion transport, transcriptional regulation, and QTL-based regulation—with known ST functions predominantly characterized in xian/indica rice, whereas their distribution and relevance in 3H geng/japonica germplasm remained unclear. Through KASP genotyping and haplotype analysis, these varieties were classified into 14 distinct haplotypes. ST level assessment of these SNPs revealed that OsSTL1, OsHKT1;1, RST1 and OsSKC1 variants significantly contribute to improved ST. Notably, RST1530A was identified as a pivotal factor in enhancing ST (57.1% increase in relative shoot fresh weight, p < 0.01) and occurs in only 3.7% of the 3H gene/japonica varieties versus 93.6% in xian/indica accessions. Further physiological and molecular analyses demonstrated that the RST1530A allele confers ST by attenuating repression of OsAS1 (6.97-fold induction under salt stress vs. 4.55-fold for RST1530G), thereby increasing its expression under salt stress. Evolutionary analysis suggests that the RST1530A allele in geng/japonica rice likely originated from xian/indica varieties through historical hybridization, involving introgression of a ≥68 Kb chromosomal segment (a minimum estimate based on flanking marker analysis). These varieties represent promising candidate donor parents for introducing RST1530A into geng/japonica rice breeding programs, pending further agronomic evaluation under field conditions.