Identification and Evolution of Salt-Tolerant Genes in Weedy Rice Through High-Throughput Resequencing

Weedy rice, a wild relative of cultivated rice, is highly stress-resistant and proliferates in paddy fields. In this study, 353 weedy rice accessions were analyzed to identify salt-tolerance genes using population evolution analysis, phenotypic screening, genome-wide association studies (GWAS), transcriptome analysis, haplotype characterization, gene knockout experiments, and Na⁺ and K⁺ ion flux assays. Population structure analysis classified the accessions into six distinct groups. Three salt-tolerant accessions-HW131, HW136, and HW119-were identified based on leaf rolling degree (LRD), leaf withering degree (LWD), chlorophyll content (ChlC), and nitrogen content (NC) traits. GWAS and transcriptome data pinpointed LOC_Os06g39270 and LOC_Os06g11860 as candidate salt-tolerance genes. Haplotype analysis and qPCR confirmed two major haplotypes: AHap2 and BHap1. A 2-bp deletion (TC) at position 818 bp in LOC_Os06g11860 was associated with severe salt sensitivity (phenotypic grade 7), whereas the wild-type exhibited strong tolerance (grade1). Knockout mutants exhibited significantly increased Na⁺ and K⁺ flux across mesophyll cell membranes compared to wild-type plants, validating LOC_Os06g11860 (OsERFH1) as a crucial salt-tolerance gene. This study provides novel genetic insights into salt-stress adaptation in weedy rice, paving the way for breeding enhanced salt-tolerant varieties.