A cation diffusion facilitator, GmCDF1, negatively regulates salt tolerance in soybean

Salt stress is one of the major abiotic factors that affect the metabolism, growth and development of plants, and soybean [Glycine max (L.) Merr.] germination is sensitive to salt stress. Thus, to ensure the successful establishment and productivity of soybeans in saline soil, the genetic mechanisms of salt tolerance at the soybean germination stage need to be explored. In this study, a population of 184 recombinant inbred lines (RILs) was utilized to map quantitative trait loci (QTLs) related to salt tolerance. A major QTL related to salt tolerance at the soybean germination stage named qST-8 was closely linked with the marker Sat_162 and detected on chromosome 8. Interestingly, a genome-wide association study (GWAS) identified several single nucleotide polymorphisms (SNPs) significantly associated with salt tolerance in the same genetic region on chromosome 8. Resequencing, bioinformatics and gene expression analyses were implemented to identify the candidate gene Glyma.08g102000, which belongs to the cation diffusion facilitator (CDF) family and was named GmCDF1. Overexpression and RNA interference of GmCDF1 in soybean hairy roots resulted in increased sensitivity and tolerance to salt stress, respectively. This report provides the first demonstration that GmCDF1 negatively regulates salt tolerance by maintaining K+-Na+ homeostasis in soybean. In addition, GmCDF1 affected the expression of two ion homeostasis-associated genes, salt overly sensitive 1 (GmSOS1) and Na+/H+ exchanger 1 (GmNHX1), in transgenic hairy roots. Moreover, a haplotype analysis detected ten haplotypes of GmCDF1 in 31 soybean genotypes. A candidate-gene association analysis showed that two SNPs in GmCDF1 were significantly associated with salt tolerance and that Hap1 was more sensitive to salt stress than Hap2. The results demonstrated that the expression level of GmCDF1 was negatively correlated with salt tolerance in the 31 soybean accessions (r = -0.56, P < 0.01). Taken together, these results not only indicate that GmCDF1 plays a negative role in soybean salt tolerance but also help elucidate the molecular mechanisms of salt tolerance and accelerate the breeding of salt-tolerant soybean. Author summary Soil salinity can seriously threaten soybean growth and development and seed germination is a key phase in the soybean growth cycle. Thus, understanding the genetic mechanism of salt tolerance at the germination stage is very important for improving the salt tolerance of soybean at the germination stage. An analysis combining linkage mapping, GWAS and whole-genome sequencing of two soybean accessions revealed that the GmCDF1 was associated with soybean salt tolerance. The transformation of soybean hairy roots and an inductively coupled plasma optical emission spectrometry (ICP-OES) analysis confirmed that GmCDF1 negatively regulates salt tolerance by maintaining ion homeostasis in soybean. A haplotype analysis found ten haplotypes of GmCDF1 in 31 soybean accessions, and a candidate gene association analysis identified two probable causative GmCDF1 polymorphisms. Moreover, higher GmCDF1 expression resulted in salt sensitivity. Our results not only reveal the role of GmCDF1 in soybean adaptation to salt stress but also help elucidate the genetic molecular mechanisms of salt tolerance and facilitate the implementation of marker-assisted selection for salt tolerance in soybean breeding programs.