3.4.5. Awn Length (AL)

Five QTLs (*qAL1.1, qAL4.1, qAL4.2, qAL8.1* and *qAL11.1*) related to awn length (AL) were detected on chromosome 1, 4, 8 and 11. Twelve CSSLs (RZ21, RZ6, RZ4, RZ62, RZ63, RZ70, RZ68, RZ119, RZ4, RZ141, RZ88 and RZ142) had ALs of 30 ± 4 mm, while the AL of 93-11 was 14 ± 3 mm (Figure 4C). The overlapping segments of RZ21, RZ6 and RZ4 were found near the molecular marker C1-7 on chromosome 1 and named *qAL1.1*. The QTLs *qAL4.1* and *An-1*/allele were detected in RZ62 and RZ63 near the molecular marker C4-8–C4-10 on chromosome 4. [40] Two other QTLs, *qAL4.2* and *An-2*/allele were detected in the overlapping segments of RZ70 and RZ68 near the molecular marker C4-19 on chromosome 4 [41]. The grains of common wild rice have long and spiny awns. In contrast, cultivated rice species have no awns or short awns with smooth surfaces (Figure S4C). The overlapping segments of RZ135 and RZ4 were located near the molecular marker C8-11 on chromosome 8 and named *qAL8.1*. We found *qAL11.1* in the overlapping segments of RZ142, RZ88 and RZ141 near the molecular marker C11-2 on chromosome 11 (Table 3; Figure S6).

### 3.4.6. Grain Shattering (SH) and Cold Tolerance (CT)

The grains of 93-11 did not shatter in either season, but the grains of five CSSLs (RZ74, R75, RZ76, RZ8 and RZ88) did shatter (Figure 5). We found *qSH4.1* in the overlapping segments of RZ74, R75 and RZ76 near the molecular marker C4-22 on chromosome 4, the *SH4* was also in the same chromosomal position [42]. *qSH11.1* in the overlapping segments of RZ8 and RZ88 (Table 3; Figure S6).

Seven QTLs (*qCT1.1*, *qCT2.1*, *qCT3.1*, *qCT5.1*, *qCT6.1*, *qCT10.1* and *qCT12.1*) related to cold tolerance (CT) were detected on chromosome 1, 2, 3, 5, 6, 10 and 12. Seventeen CSSLs (RZ10, RZ11, RZ12, RZ34, RZ40, RZ41, RZ85, RZ86, RZ87, RZ99, RZ100, RZ101, RZ136, RZ137, RZ158, RZ162 and RZ163) were more tolerant to cold stress compared with 93-11 (Figure S3). We identified *qCT1.1* in the overlapping segments of RZ10, RZ11 and RZ12 near the marker C1-16 on chromosome 1, which also contained the gene *OsRAN1* [43]. *qCT2.1* was found in the overlapping segments of RZ34, RZ40, RZ137, RZ158 and RZ170 near the marker C2-19. *qCT3.1* was detected in the overlapping segments of RZ40 and RZ41 near marker C3-2. *Qc T5*.1 was identified in the overlapping segments of RZ85, RZ86, RZ87 and RZ170 near the marker C5-20 on chromosome 5, the *OsiSAP8* was also in the same chromosomal position [44]. The QTLs *qCT6.1* was found in the overlapping segments of RZ99, RZ100, RZ101 and RZ171 near the marker C6-20 on chromosome 6, the *OsPYL9* was also in the same chromosomal position [45]. chromosome 10 contained the QTL *qCT10.1*, which was identified in the overlapping segments of RZ136, RZ137 and RZ171 near the marker C10-3. The overlapping segments of RZ158, RZ162 and RZ163 were located near the marker C12-11 on chromosome 12 and named *qCT12.1* (Table 3).

**Figure 5.** Phenotypic variation of grain shattering in RZ8 and 93-11; Whole plant phenotype showing grain shattering (bar = 50 cm), grain shattering (bar = 10 cm, red arrows).

### 3.4.7. Identification of Loci Related to Quality Traits

In five CSSLs (RZ5, RZ112, RZ19, RZ75 and RZ106), significant variation with 93-11 in LSC, LMC, AC, SC, GC and SCC (Figures S4 and S5). Segments of these CSSLs are located in chromosomes 1, 4, 7 and 11. Six related trait genes (*LSC11.1*, *LMC11.1*, *AC1.1*, *SC1.1*, *GC4.1* and *SCC7.1*) were identified; *AC1.1* and *SC1.1* may be the same gene. *AC1.1* and *SC1.1* may be the allele gene of *A. GC4.1*, SCC7.1 and *Bh4, Rc* were in the same chromosome position [46–48].

### *3.5. Construction of a Secondary Population and Mapping of qCT2.1*

Our results showed that there was no significant difference in the appearance of RZ34 and 93-11 seedlings before cold treatment (Figure 6A). RZ34 had a lesser degree of wilting after cold treatment compared with 93-11 (Figure 6B). Furthermore, 93-11 died three days after the cold treatment, while RZ34 survived and continued to grow (Figure 6C). The results show that RZ34 seedlings were more tolerant to cold stress than 93-11. All the F1 individuals in the RZ34 line exhibited a cold tolerant phenotype. We selected some of the F1 plants and the related molecular markers showed that these plants were heterozygous for the tested genotypes. We obtained a total of 311 plants in the F2 population, among them 223 plants were cold tolerant, and 88 plants were susceptible to cold stress. This represented a 3:1 segregation ratio for cold tolerance which consistent with Mendelian's rules (χ<sup>2</sup> = 1.630 < χ20.05,1 = 3.84), demonstrating that this cold tolerance trait was controlled by a single dominant QTL.

We identified the QTL *qCT2.1* in the segmen<sup>t</sup> of RZ34 (Figure 7A) between markers JM2-3 (21.7 Mb) and C2-22 (29.18 Mb). Based on the tracking and overlap of three CSSLs (RZ37, RZ38 and RZ39) segments, the range of *qCT2.1* was reduced to 4.47 Mb (Figure 7A). Five InDel markers (dxw-3, dxw-9, dxw-8, dxw-5 and dxw-4) were developed between C2-18 and C2-20 (Table S4). In order to reduce phenotypic identification error and improve the accuracy of QTL mapping, we selected individual

plants with extreme phenotypic values to detect genotypes. We identified the phenotypes of 311 plants in the F2 population. Among them, the genotypes of 56 cold-tolerant plants (scale 9–7) and 22 cold-sensitive plants (scale 0) were detected for linkage analysis. Five recombinant plants (R1, R2, R3, R4 and R5) were selected from the constructed F2 population (Table S5). These plants confirmed that *qCT2.1* is located in a 1.7 Mb region between molecular markers dxw-4 and dxw-9 (Figure 7B) and that the LOD value of *qCT2.1* was 8 (Figure 7C).

**Figure 6.** Cold stress treatment to RZ34 and 93-11 at seedling stage. (**A**) Before cold stress (bar = 10 cm); (**B**) after cold stress (bar = 10 cm) and (**C**) after three days of recovery (bar = 10 cm).

**Figure 7.** Mapping of grain-shattering QTL *qCT2.1*, (**A**) QTL analysis by overlapping segments of recombinants RZ34, RZ35 and RZ29, blue intervals indicate DP30 genotype; blank intervals indicate: 93-11 genotype; shadow intervals indicate heterozygous genotype; (**B**) linkage analysis of *qCT2.1* yellow intervals indicate RZ34 genotype, blank intervals indicate 93-11 genotype, shadow intervals indicate heterozygous genotype; (**C**) LOD value of *qCT2.1.* dxw-3, dxw-8, dxw-9, dxw-5 and dxw-4 are InDel markers.
