**2. Results**

## *2.1. Breeding Scheme for Development of Advanced Plant Material*

We introgressed the salt-tolerance *hst1* gene from "Kaijin" into the genetic background of the high-yielding "Yukinko-mai" by three backcrosses followed by two rounds of self-fertilization (Figure 1A). To accelerate the breeding cycle, we used a biotron speed-breeding system, with controlled temperature and daylength, restriction of tillers, and embryo rescue (Figure S1). At each cross, the plants produced a good quantity of fertilized seed; the cross success rate ranged between 54% and 69% (Table S1), and seeds from three or four plants were sufficient to develop new progeny. Each advanced generation took approximately 70 days from germination to flowering and 10 days from pollination to embryo rescue (Figure S1). The total duration of each generation varied according to days to flowering. Using this speed-breeding technique, we developed the BC3F<sup>3</sup> population, carrying our desired allele in the homozygous state, in six generations and 17 months. *Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 4 of 22

**Figure 1.** Single nucleotide polymorphism (SNP) marker-aided introgression of *hst1* from "Kaijin" into "Yukinko-mai". (**A**) The *hst1* gene was transferred from highly salt-tolerant "Kaijin" into "Yukinko-mai". "Kaijin" was backcrossed to "Yukinko-mai" (WT) 3 times followed by 2 rounds of self-pollination. The table shows the selection results at each generation: T, total number of tested plants; G/G, number of plants not carrying donor allele; G/A, number of plants carrying donor allele in heterozygous state; A/A, plants carrying donor allele in homozygous state. The number after "#" is the individual plant number used in backcrossing or self-pollination. (**B**) Advanced breeding individuals were genotyped by direct sequencing. The red box represents nucleotide 1975 of the *OsRR22* locus, which is responsible for salt tolerance. **Figure 1.** Single nucleotide polymorphism (SNP) marker-aided introgression of *hst1* from "Kaijin" into "Yukinko-mai". (**A**) The *hst1* gene was transferred from highly salt-tolerant "Kaijin" into "Yukinko-mai". "Kaijin" was backcrossed to "Yukinko-mai" (WT) 3 times followed by 2 rounds of self-pollination. The table shows the selection results at each generation: T, total number of tested plants; G/G, number of plants not carrying donor allele; G/A, number of plants carrying donor allele in heterozygous state; A/A, plants carrying donor allele in homozygous state. The number after "#" is the individual plant number used in backcrossing or self-pollination. (**B**) Advanced breeding individuals were genotyped by direct sequencing. The red box represents nucleotide 1975 of the *OsRR22* locus, which is responsible for salt tolerance.

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 BC3F2 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 BC3F2 genome was "Kaijin" homozygous and 7.6% remained unfixed as heterozygous

To investigate the genetic similarities between our advanced line and the parents, we analyzed BC3F2 #31-2-4 using whole-genome sequencing. After we filtered out low-reliability SNPs/indels, #31-

*2.3. Recovery Rate and Characterization of BC3F2 #31-2-4 Genome* 

(Table 1).
