*2.5. Fine-Mapping of qDOM3.1 for Seed Dormancy*

To further fine map *qDOM3.1*, we selected the heterozygous lines in the CSSL-derived F2 population flanked by the markers RM14238 and MP030012, and self-pollinated these heterozygous lines to generate a larger segregating population (*n* = 2500). Through genotyping with 14 additional markers, seven informative recombinants with *qDOM3.1* were identified. A progeny test of the informative recombination plants delimited *qDOM3.1* for seed dormancy to a 90 kb region (Figure 5 and Figure S3, Supplementary Materials). This region encompassed 19 open reading frames (ORFs) according to the RGAP database (available online: http://rice.plantbiology.msu.edu/, Release 7).

**Figure 5.** Fine mapping of *qDOM3.1*. The QTLs narrowed down to the region flanked by MP97-8-1 and MMP4-1 on the upper end of chromosome 3. Some important recombinant plants derived from a large F2 group generated by selfing a single individual heterozygous at the *qDOM3.1* region and divided into 7 groups based on their genotypes. Gmax (mean ± SD) (%) is given on the right for each genotype. The phenotypes of each recombinant individual were evaluated by germination experiments. \*\* Indicates significant difference at *p* < 0.01 by Dunnett's test against the control.

The 19 genes included 11 expressed proteins, 1 transposon protein, 2 retrotransposon proteins, 1 hypothetical protein, and 4 genes with functional annotation. The chromosomal synteny analysis between NIP and ZS97 showed there were 7 genes missing in the ZS97 (available online: http: //rice.hzau.edu.cn/cgi-bin/gb2/gbrowse\_syn/3rice\_syn/) (Figure 6). As the candidate gene should be dominant in ZS97 (Figure 3), those 7 genes were ruled out from the candidate genes. Thus, only 12 genes remained, including 10 expressed proteins, 1 gene annotated as tubulin/FtsZ domain-containing protein (LOC\_Os03g01530), and another annotated as DNA-binding protein (LOC\_Os03g01540). According to the expression profile in the database (http://rice.plantbiology.msu.edu/expression.shtml), 4 genes (LOC\_Os03g01430, LOC\_Os03g01450, LOC\_Os03g01460, and LOC\_Os03g01520) had no expression or very low expression among all the tissues. Therefore, those 4 genes were unlikely to be our candidate genes, leaving 8 genes as candidate genes. The expression profiles were obtained from the RiceXPro website (available online: http://ricexpro.dna.affrc.go.jp/) (Figure S4, Supplementary Materials). None of the 8 genes were seed-specific expressed, except LOC\_Os03g01360 that had a relatively higher expression in embryo from 7 days after flowering until 42 days after flowering. Based on the sequence comparison between NIP and ZS97, only LOC\_Os03g01530 had no amino acid change in the coding region; all the other 7 genes contained at least one missense variant.

**Figure 6.** Chromosomal synteny analysis of ZS97 and NIP of candidate region on chromosome 3. The genes in which ZS97 was not contained are indicated with the final three numbers of the gene ID (e.g., LOC\_Os03g01365 is displayed as 365). In NIP panel, green gene symbol means gene direction from left to the right and orange gene symbol means gene direction from right to the left.
