*2.3. Recovery Rate and Characterization of BC3F<sup>2</sup> #31-2-4 Genome*

To investigate the genetic similarities between our advanced line and the parents, we analyzed BC3F<sup>2</sup> #31-2-4 using whole-genome sequencing. After we filtered out low-reliability SNPs/indels, #31-2-4 showed 118 454 SNPs/indels, comprising 106 288 "Yukinko-mai"-type homozygous alleles, 3157 "Kaijin"-type homozygous alleles, and 9009 heterozygous alleles (Table 1). These SNPs/indels lie across the genome with deep coverage (Figure 2, dots), indicating high resolution and successful genome-wide genotyping. Allele types formed dense blocks on chromosomes (Figure 2, vertical bars), clearly showing recovered regions ("Yukinko-mai" homozygous blocks), "Kaijin" genome segments ("Kaijin" homozygous blocks), and unfixed segments (heterozygous blocks) (Figure 2, horizontal bar). "Yukinko-mai" chromosomes (Chrs.) 5, 11, and 12 were recovered almost completely. There were small "Kaijin" segments in Chrs. 1, 4, 7, 9, and 10, large "Kaijin" segments in Chrs. 2, 3, and 6, small heterozygous segments in Chr. 4, and large heterozygous segments in Chrs. 3, 6, 8, and 9. Interestingly, we identified some genotype blocks overlapping other genotype blocks (Figure 2, chr08, 5–10 Mb; chr09, 10–12 Mb), resulting from continuous recombination events in these extremely short regions [50,51]. We calculated the genome recovery rate from the number of "Yukinko-mai" alleles out of the total number; the BC3F<sup>2</sup> genome recovered 93.5% of the "Yukinko-mai" genome, from 89.7% homozygous alleles and 7.6% heterozygous alleles (Table 1; Section 4.7). This score is close to the theoretical value of 93.7% following three backcrosses and one self-fertilization. In addition, 2.7% of the BC3F<sup>2</sup> genome was "Kaijin" homozygous and 7.6% remained unfixed as heterozygous (Table 1).


**Table 1.** Single nucleotide polymorphism (SNP)/indel detection in BC3F<sup>2</sup> #31-2-4. SNPs/indels in BC3F<sup>2</sup> #31-2-4 are classified into "Yukinko-mai"-type (homo[zygous]), "Kaijin"-type (homo(zygous)), and Hetero(zygous) alleles.

To estimate the effects of variants on phenotypes, we listed variants causing protein sequence alterations (e.g., frameshift and in-frame indels, non-synonymous SNPs, SNPs/indels at splice donor/acceptor sites etc.) in SnpEff software for Sequence Ontology and effect prediction (Figure 3). In BC3F2, 207 "Kaijin" homozygous and 536 heterozygous variants were found in exon and splice sites. Only 71 and 230, respectively, of those caused protein sequence alterations (Figure 3; Table S2). These non-synonymous changes occurred in only four genes for agronomic traits (Table 2).

Hetero(zygous) alleles.

To estimate the effects of variants on phenotypes, we listed variants causing protein sequence alterations (e.g., frameshift and in-frame indels, non-synonymous SNPs, SNPs/indels at splice donor/acceptor sites etc.) in SnpEff software for Sequence Ontology and effect prediction (Figure 3). In BC3F2, 207 "Kaijin" homozygous and 536 heterozygous variants were found in exon and splice sites. Only 71 and 230, respectively, of those caused protein sequence alterations (Figure 3; Table S2).

These non-synonymous changes occurred in only four genes for agronomic traits (Table 2).

**Table 1.** Single nucleotide polymorphism (SNP)/indel detection in BC3F2 #31-2-4. SNPs/indels in BC3F2 #31-2-4 are classified into "Yukinko-mai"-type (homo[zygous]), "Kaijin"-type (homo(zygous)), and

> 'Yukinko-mai" (homo) 84 927 21 361 106 288 89.7 "Kaijin" (homo) 2329 828 3157 2.7 Hetero 7556 1453 9009 7.6 Total 94 812 23 642 118 454 100.0

**Genotype Number of Variants Genome % SNPs Indels Total** 

**Figure 2.** Positions and depths of SNPs/indels and genotype blocks on BC3F2 #31-2-4 genome**.** Dots show coverage depth of SNPs/indels. Vertical bars show their positions by genotype (color). Horizontal bar shows densities of SNPs/indels densities in 10,000-nt sliding window by genotype **Figure 2.** Positions and depths of SNPs/indels and genotype blocks on BC3F<sup>2</sup> #31-2-4 genome. Dots show coverage depth of SNPs/indels. Vertical bars show their positions by genotype (color). Horizontal bar shows densities of SNPs/indels densities in 10,000-nt sliding window by genotype (colour). Black, "Yukinko-mai"-type homozygous; red, "Kaijin"-type homozygous; blue, heterozygous. Green bars represent chromosome length.
