*2.3. SNP Allele Calling*

Genotype calling was performed in order to identify SNPs that originated from the *indica* and *japonica* genomes. There were two types of values calculated in this study: variety value and reference value. The variety value calculated whether it was of *japonica*-type parental allele (Yukara allele) of Tongil or a Tongil-like variety or not. The variety value of SNP was calculated as the sum of the following values: '1' (IR 8 allele); '2' (TN1 allele); '4' (Yukara allele); and '0' (all others). If the SNP is the same as the Nipponbare reference, the value '1' was given to the SNP; otherwise, the value '0' was given. The SNPs showing variety value '4' and reference value '1' were called *japonica*-type SNPs. Then, the total number of *japonica*-type SNPs in each 100 kb block, which is the approximate chromosomal distance for the linkage disequilibrium (LD) decay rate in rice [28], in each chromosome was counted to identify the introgressed regions of *japonica*.

### *2.4. SNP Marker Development for the Fluidigm Platform*

A total of 39 representative genes were selected from the selected regions. The representative genes were well-annotated in the public gene/QTL databases, as of 6 January 2017 (RAP and UniProt). Then, only the genes containing non-synonymous SNPs in the predicted exon and UTR regions between HYVs and non-HYVs were selected. We assumed that these genes might be a part of the candidate genes that are associated with *japonica*-originated traits. Out of the many SNPs in the genes, only one SNP with a substitution polymorphism between *indica* (IR 8 and TN1) and *japonica* (Nipponbare and Yukara) per representative gene was selected for the genomic validation. The SNP markers for the Fluidigm platform (Fluidigm Corporation, San Francisco, CA, USA) were designed using the method by Seo et al. [29]. To design Fluidigm SNP genotyping assays, 60–150 bp sequences, flanking the selected SNPs on either side, were aligned by BLAST. Finally, the selected SNPs and flanking sequences were uploaded on the D3 Assay Design website [30]. After confirming the results, the designed assays were ordered. One Fluidigm SNP assay contained the Allele-Specific Primer 1 (ASP1), ASP2, Locus-Specific Primer (LSP), and Specific Target Amplification (STA) primer.
