Integrative 16S rRNA and transcriptome analysis reveals the molecular mechanisms underlying salt- tolerant germination in highland barley (Hordeum vulgare var. coeleste Linnaeus) seeds

Salt stress is a critical abiotic factor that impairs crop seed germination and limits agricultural productivity. Elucidating the mechanisms governing salt tolerance is essential for development of salt-tolerant crop varieties. In this investigation, 217 accessions of highland barley (<i>Hordeum vulgare</i> var. <i>coeleste Linnaeus</i>) were evaluated. Germination assays conducted under 200 mmol/L and 500 mmol/L NaCl conditions identified a salt-tolerant variety 37 and a salt-sensitive variety 44. By integrating transcriptome sequencing, 16S rRNA sequencing, and Na⁺/K⁺ content analysis, we systematically investigated the molecular mechanisms underlying salt-tolerant germination in highland barley seeds. Our findings revealed that the salt-tolerant variety 37 maintained a high germination rate of 98% under 500 mmol/L NaCl stress, with lower Na⁺ accumulation (4.24 g/kg) and a lower Na⁺/K⁺ ratio (2.59) compared to the salt-sensitive variety 44 (Na⁺ accumulation: 4.89 g/kg, Na⁺/K⁺ ratio: 3.62). Analysis of 16S rRNA sequencing data showed a significant increase in the abundance of the endophytic bacterium <i>Brevundimonas</i> in salt-tolerant variety 37 under high-salt conditions, which was positively correlated with K⁺ content. In contrast, the dominant bacterium <i>Rhodococcus</i> in salt-sensitive variety 44 exhibited a positive correlation with Na⁺ content and the Na⁺/K⁺ ratio. Transcriptome sequencing identified 1,467 and 1,644 differentially expressed genes (DEGs) in salt-tolerant variety 37 and salt-sensitive variety 44, respectively. Pathway enrichment analysis indicated that DEGs in salt-tolerant variety 37 were primarily associated with "potassium ion homeostasis" and "response to oxidative stress". Weighted gene co-expression network analysis (WGCNA) identified 5 co-expression modules, among which the MEyellow module was correlated with Na⁺ content (r = 0.59). Ten core genes were identified, including WRKY transcription factor (<i>HORVU.MOREX.r3.3HG0268090</i>) and receptor protein kinase (RPK; <i>HORVU.MOREX.r3.4HG0331910</i>). A total of 174 <i>HvRPK</i> genes were identified, distributed across 7 chromosomes with a predominant localization on chromosome 2. These genes exhibited functional conservation and were involved in salt stress signaling pathways. Phylogenetic, collinearity, and cis-element analyses further supported their regulatory role in salt stress responses. This study clarifies the key mechanisms underlying salt-tolerant germination in highland barley seeds, providing valuable insights and genetic resources for the molecular breeding of salt-tolerant crops.