*2.2. Introgression of hst1 into "Yukinko-mai" (WT) and Genotyping of Advanced Progeny Using SNP Marker*

We used SNP-based genotyping by Sanger sequencing to identify plants harboring the donor allele in each breeding round. A SNP in the *OsRR22* (*hst1*) gene confers the salinity tolerance of the donor parent "Kaijin". The breeding lines were selected on the basis of target peaks of G/A heterozygosity (nucleotide 1975 of the *OsRR22* locus) in the F1, BC1F1, BC2F1, and BC3F<sup>1</sup> generations and of A/A homozygosity at the same locus in the BC3F<sup>2</sup> generation (Figure 1B). In the F<sup>1</sup> to BC3F<sup>1</sup> generations, we obtained two genotypes: either homozygous, lacking the donor allele (G/G), or heterozygous (G/A) (Figure 1A). The heterozygous BC3F<sup>1</sup> population was self-pollinated to develop BC3F<sup>2</sup> lines that carried the donor allele in the homozygous state (A/A). BC3F<sup>2</sup> plants (A/A) morphologically similar to the recurrent parent were self-pollinated to develop the BC3F<sup>3</sup> generation. We sequenced the whole genome of the BC3F<sup>2</sup> line #31-2-4 to compare it with the parental genome, and characterized it.
