Single candidate gene for salt tolerance of Vigna nakashimae (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 can- didate gene for salt tolerance in Vigna nakashimae, one of the coastal species in the genus Vigna. Using in- traspecific variation, we performed a forward genetic analysis and identified a strong QTL region harboring similar to 200 genes. To further narrow down the candidate genes, we performed a comparative transcriptome analy- sis, using the genome sequence of azuki bean (V. angularis) as a reference. However the detected differen- tially-expressed genes (DEGs) did not include those related to salt tolerance. As we suspected that the target gene in V. nakashimae is missing in V. angularis, we sequenced the whole genome sequence of V. nakashimae with long-reads. By re-analyzing the transcriptome data with the new reference genome, we successfully identified POCO1 as a candidate gene, which was missing not only in V. angularis but also in the salt-sensi- tive accession of V. nakashimae. 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.