*2.4. QTL Analysis of Low-Temperature Germinability*

After combining the bin-map and phenotyping data of each RIL line, scanning of the LTG QTL was carried out. A total of 11 QTLs were identified on chromosome 1 (11.48–19.34 Mb), chromosome 3 (0.00–1.31 Mb), chromosome 4 (6.10–11.41 Mb), chromosome 6 (0.34–0.74 Mb), chromosome 7 (6.98–9.21 Mb; 18.93–21.76 Mb), chromosome 9 (5.91–6.83 Mb; 14.91–21.38 Mb), chromosome 10 (1.20–1.60 Mb), and chromosome 12 (0.86–2.35 Mb; 21.05–25.16 Mb). The confidence intervals associated with these QTLs spanned the genomic physical position from 0.07 to 7.86 Mb compared with the reference genome of rice (Table 2, Figure 3A). Five QTLs showed relatively small confidence intervals of less than 1.5 Mb, including *qLTG-3* (1.31 Mb), *qLTG6* (0.40 Mb), *qLTG-9a* (0.92 Mb), *qLTG-10* (0.40 Mb), and *qLTG-12a* (1.49 Mb). Among these QTLs, phenotypic variation ranged from 10.30% to 21.04% (Table 2, Figure 3B). Overall, five QTLs depicted the explained phenotypic variation exceeding 15%, and two major QTLs were detected: *qLTG7b*, found on chromosome 7, with 21.04% phenotypic variation and 7.39 of the LOD value; and *qLTG10*, located on chromosome 10 and comprised of 21.00% phenotypic variation with a 7.38 LOD value. Furthermore, eight QTL alleles, *qLTG1*, *qLTG3*, *qLTG6*, *qLTG7a*, *qLTG7b*, *qLTG9a*, *qLTG10*, and *qLTG12a*, had a positive effect inherited from LTH. The other three QTL alleles, *qLTG4*, *qLTG9b*, and *qLTG12b*, had a positive effect inherited from SN265 (Figure 3B). Based on the high-resolution bin map, two QTLs, *qLTG3* and *qLTG7b*, were localized to the chromosome intervals that subsumed the cloned LTG genes. The QTL named *qLTG3* was located in the interval of the LTG gene described as *qLTG3-1*, with a 4.71 LOD value and 14.02% phenotype variance [9]. The QTL named *qLTG7b* had a 7.39 LOD value and 21.04% phenotypic variation and was located on *OsSAP16*, a rice LTG gene that has already been identified by the association study method [10]. PCR amplification and sequencing were conducted to screen out the causal polymorphisms of *qLTG3-1* and *OsSAP-16*, which determined that LTH has two cold-tolerant genes (Figure 3E,F). Additionally, these results also indicate that the data used for scanning QTLs are entirely effective (Figure 3D,E,G,H).



Chr., chromosome; Positions in linkage map (unit: Mb); Logarithm (base 10) corresponding QTL peak; Percentage phenotypic the corresponding QTL;e Additive effect of the corresponding QTL.
