*2.1. Population Sequencing and Linkage Map Construction*

We used a strategy of sequencing-based map construction to conduct QTL mapping for the BIL derived from the cross between Sasanishiki and Habataki (Figure 1A). We sequenced a segregating population of Sasanishiki and Habataki BILs together with parental lines on an Illumina HiSeq2500 platform. A total of 224.02 GB of raw data were generated for all of the BILs, with approximately 6.29-fold depth for each BIL, 22.94 GB for Habataki (51.00-fold), and 23.24 GB for Sasanishiki (56.00-fold). We aligned the sequence data to the reference genome (Os-Nipponbare-Reference-IRGSP-1.0) using SOAP2 software [10,11]. A total of 1,947,668 single-nucleotide polymorphisms (SNPs) with homozygous genotypes between both parents were identified using the SOAPsnp software [12]. These SNPs were used as potential markers in the subsequent analysis. The SNP markers localized to highly repetitive regions, and those with low genotyping scores were removed to avoid ambiguity in linkage map construction. We used an effective imputation model, k-nearest neighbor algorithm to impute the missing genotypes of each RIL caused by low-coverage sequencing [13]. Finally, we used 1,591,495 high-quality polymorphic SNP markers to construct a recombinant bin map (Figure 1B). Subsequently, a recombinant bin map was constructed, and the map contained 3652 recombinant blocks, with the average genetic length of 0.44 cM. Then, we determined the introgression rate of the Habataki pedigree using the data of bin map. The introgression rate showed a normal distribution that indicated that the population is ideal for the subsequent survey (Figure 1C). We analyzed the correlation between

the introgression rate of Habataki and the important yield-related traits. The results showed that the introgression rate was significantly negatively correlated to 1000-grain weight (TGW) (Figure 1D).

**Figure 1.** Schematic overview of the parent backcross recombinant inbred line (BIL) system construction and the map of genome-wide graphic genotypes. (**A**) The technology roadmap using genome sequence and quantitative trait locus (QTL) analysis of BILs and BILF1s to uncover heterosis-related loci for yield increase. (**B**) Graphic genotypes of 85 BILs were identified by a sliding window approach along each chromosome. Various colors represent different genotypes. (**C**) The introgression rate of the Habataki pedigree among the BILs. (**D**) The correlation between the introgression rate of the Habataki pedigree and yield-related traits. The dotted lines indicate significance at the 5% level.
