Single candidate gene for salt tolerance of (Ohwi) Ohwi & Ohashi identified by QTL mapping, whole genome sequencing and triplicated RNA-seq analyses.

Salt tolerance has been an important issue as a solution for soil salinization and groundwater depletion. To challenge this issue, genetic diversity of wild plants must be harnessed. Here we report a discovery of a candidate gene for salt tolerance in , one of the coastal species in the genus . Using intraspecific variation, we performed a forward genetic analysis and identified a strong QTL region harboring ~200 genes. To further narrow down the candidate genes, we performed a comparative transcriptome analysis, using the genome sequence of azuki bean () as a reference. However the detected differentially-expressed genes (DEGs) did not include those related to salt tolerance. As we suspected that the target gene in is missing in , we sequenced the whole genome sequence of with long-reads. By re-analyzing the transcriptome data with the new reference genome, we successfully identified as a candidate gene, which was missing not only in but also in the salt-sensitive accession of . Further comparative analysis revealed that the tolerant genotypes conserved the ancestral form of the locus, while the sensitive genotypes did not. We also emphasize the pitfalls in our study, such as position effect in a growth chamber, missing important genes in the reference genome, and limited reproducibility of RNA-seq experiments.