*2.1. Phenotypic Variation among the Parent and RIL Populations*

After incubating at 28 ◦C for three days, the germination rates of LTH and SN265 were 100%, indicating that both parents share a similar germination rate at a normal temperature, as presented in Figure 1A. However, when they were incubated at 15 ◦C for six days to determine their low temperature germinability, LTH and SN265 showed broad differences in their germination rates, as shown in Figure 1A,B. There was a delay of about two days in germination after incubating LTH under low temperature conditions, and, thereafter, the germination rate reached up to 90% after five days of incubation. Comparatively, for SN265, germination started after five days of incubation and, thereafter, took five more days to reach 90% of the germination of LTH, which shows a clear difference in the time taken for germination by LTH and SN265 (Figure 1B). A range of five to eight days was chosen for a proper and dynamic comparison of the low-temperature germinability among the parents and RILs (Figure 1C–F). At all four time points, the distributions of the germination percentages of the RIL populations were continuous, indicating that low-temperature germinability is controlled by QTLs. Generally, it is already known that the longer the germination time, the higher the germination percentage. Therefore, on the basis of the larger differences in germination between the parents after six days (LTH and SN265 showed 86.5% and 15.4%, respectively), those data were used for the subsequent QTL mapping.

**Figure 1.** The seed germination of two parents and their recombinant inbred lines (RILs) at normal (28 ◦C) and low (15 ◦C) temperatures. (**A**). The germination phenotype of the two parents, Lijiangxintuanheigu (LTH) and Shennong265 (SN265), as well as the four RIL lines incubated for 3 days at 28 ◦C and for 6 days at 15 ◦C. R136 and R145 have 11 positive QTLs, whereas R77 and R144 have no positive QTLs. The bars = 5 mm; (**B**). The germination behavior of LTH (black) and SN265 (red) under low temperatures at 15 ◦C. The means are shown in triplicate; (**C**–**F**). The frequency distribution of low-temperature germinability in the RILs incubated for 5 days (**C**), 6 days (**D**), 7 days (**E**), and 8 days (**F**). Arrowheads indicate LTH or SN265.

#### *2.2. Bin Map Construction and Comparison of the Physical Map to the Genetic Map*

For proper identification of the SNP between the two parents as molecular markers, deep resequencing was done for LTH and SN265. The effective sequencing depths of LTH and SN265 were about 19-fold and 17-fold, respectively. After resequencing, a total of 123,859 SNPs were produced between the two parents. Construction of the genetic linkage map for the RILs was carried out by re-sequencing the 144 RIL lines, which were already derived from LTH and SN265. In this way, these 144 RIL lines produced a range of reads between about 7,448,879 and 12,118,209, with a mean value of 9,660,250. The overall effective depth coverage of these RIL lines ranged from 5.98-fold to 9.73-fold, with an average depth of 7.75-fold. About 58,738 recombination breakpoints were used to construct the fine bin map. Each RIL line was comprised of breakpoints ranging from 236 to 1007 breakpoints, with an average value of 405. The 144 RIL lines were merged into a high-density bin map comprising 2,818 recombination bins of the 12 rice chromosomes, including most recombination events (Figure 2A). The average of the physical intervals for the adjacent bins was observed between 15.00 kb and 3.60 Mb, with a mean value of 128.80 kb, where most of the bins with physical intervals less than 100.00 kb were found to be around 68.60%. Overall, only 32 bins exceeded a physical interval of 1.00 Mb, most of which were in centromeric regions. The average physical distance between the bin markers was 98.23 kb and 169.03 kb on chromosome 10 and chromosome 1, respectively. Then, we constructed 12 chromosomes with a total genetic distance of 2,840.12 cm (Figure 2B). The average genetic distance observed between the two markers was nearly 1.01 cm. Among all chromosomes under consideration, chromosome 1 was the longest, with 254 bin markers, and its genetic distance was 338.65 cm. In contrast, chromosome 9 was seen to be the shortest one, with 188 bin markers encompassing a genetic distance of 111.32 cm, as given in Table 1. Comparing the genetic distance between chromosome 9 and 11, the average values were 0.59 cm and 1.74 cm, respectively.

**Figure 2.** High-resolution genotyping and comparison of the physical maps and genetic maps of RILs. (**A**). Aligned recombination maps of 144 RILs from a cross between LTH and SN265. The two genotypes are indicated by blue (SN265) and red (LTH); (**B**). Comparison of the physical maps and genetic maps. Left: physical map. Right: genetic map.

#### *2.3. The Quality and Accuracy of the Bin Map*

QTL mapping for the typical panicle trait named *Dense and Erect Panicle1* (*DEP1*) was performed to estimate the accuracy and mapping resolution ability of the respective bin maps and the RILs. Careful observations were taken for the panicle curvature of the 144 RILs lines 25 days after the start of heading, whereas the erect-type panicle lines remained erect. One QTL was identified on chromosome 9, with a peak interval of 16.4 Mb, as shown in Figure 3C. The QTL peak was located on the previously characterized and cloned *DEP1*/*EP1* gene. Sequence comparisons of the *DEP1* region between LTH and SN265 illustrated the replacement of a 637-bp in the middle of exon-5 by a 12-bp sequence (Figure 3F), which caused a frame shift mutation, as described previously [28].


**Table 1.** Characteristics of the high-density genetic map derived from a cross between LTH and SN265.

<sup>a</sup> Chr., indicates chromosome; <sup>b</sup> No. markers, the number of markers on the chromosome.

**Figure 3.** (**A**). QTL scan using whole-genome sequencing and a sequence comparing the panicle curvature and low-temperature germinability QTLs near the previously identified genes. Curves indicate the chromosome locations (Mb) and the low-temperature germinability (LOD) values of the detected QTLs. Arrowheads represent the relative genetic positions of the candidate genes. (**A**). Genomic locations of the 11 QTLs with strong effects for low-temperature germinability (LOD > 3) identified in the RIL population; (**B**). Plots of the additive effect and the allele effect of the 11 QTLs. (**C**). A plot of the LOD values of the panicle curvature. (**D**). A plot of the LOD values of the low-temperature germinability on chromosome 3. (**E**). A plot of the LOD values of low-temperature germinability on chromosome 7. (**F**). Sequence comparisons of the *DEP1* region between LTH and SN265. (**G**). Sequence comparisons of the *qLTG3-1* region between LTH and SN265. (**H**). Sequence comparisons of the *OsSAP16* region between LTH and SN265.
